Compositions and methods of treating facioscapulohumeral muscular dystrophy

ABSTRACT

Disclosed herein are polynucleic acid molecules, pharmaceutical compositions, and methods for treating Facioscapulohumeral muscular dystrophy.

CROSS-REFERENCE

This application is a continuation of U.S. Application Ser. No.17/200,612, filed on Mar. 12, 2021, which claims the benefit of U.S.Provisional Application No. 62/992,071 filed on Mar. 19, 2020, and U.S.Provisional Application No. 63/066,655 filed on Aug. 17, 2020, each ofwhich is incorporated herein by reference in its entirety.

REFERENCE TO A SEQUENCE LISTING XML

This application contains a Sequence Listing which has been submittedelectronically in XML format. The Sequence Listing XML is incorporatedherein by reference. Said XML file, created on May 22, 2023, is named45532-742_302_SL.xml and is 1,928,393 bytes in size.

BACKGROUND OF THE DISCLOSURE

Gene suppression by RNA-induced gene silencing provides several levelsof control transcription inactivation, small interfering RNA(siRNA)-induced mRNA degradation, and siRNA-induced transcriptionalattenuation. In some instances. RNA interference (RNAi) provides longlasting effect over multiple cell divisions. As such, RNAi represents aviable method useful for drug target validation, gene function analysis,pathway analysis, and disease therapeutics.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY OF THE DISCLOSURE

Disclosed herein, in certain embodiments, are polynucleic acid moleculesand pharmaceutical compositions for modulating a gene associated withmuscle atrophy, especially Facioscapulobumeral muscular dystrophy(FSHD). In some embodiments, also described herein are methods oftreating muscle atrophy, especially FSHD, with a polynucleic acidmolecule or a polynucleic acid molecule conjugate disclosed herein.

Disclosed herein, in certain embodiments, is a polynucleic acid moleculeconjugate comprising an antibody or antigen binding fragment thereofconjugated to a polynucleic acid molecule that hybridizes to a targetsequence of DUX4, and the polynucleic acid molecule conjugate mediatesRNA interference against the DUX4. In certain embodiments, the antibodyor antigen binding fragment thereof comprises a non-human antibody orbinding fragment thereof, a human antibody or antigen binding fragmentthereof, a humanized antibody or antigen binding fragment thereof,chimeric antibody or antigen binding fragment thereof, monoclonalantibody or antigen binding fragment thereof, monovalent Fab′, divalentFab2, single-chain variable fragment (scFv), diabody, minibody,nanobody, single-domain antibody (sdAb), or camelid antibody or antigenbinding fragment thereof. In certain embodiments, the antibody orantigen binding fragment thereof is an anti-transferrin receptorantibody or antigen binding fragment thereof.

In certain embodiments, the polynucleic acid molecule comprises a sensestrand and/or an antisense strand, and wherein the sense strand and/orthe antisense strand each independently comprises at least one 2′modified nucleotide, at least one modified internucleotide linkage, orat least one inverted abasic moiety. In certain embodiments, thepolynucleotide hybridizes to at least 8 contiguous bases of the targetsequence of DUX4. In certain embodiments, the polynucleotide is fromabout 8 to about 50 nucleotides in length or from about 10 to about 30nucleotides in length. In certain embodiments, the polynucleic acidmolecule comprises a sense strand and/or an antisense strand, and thesense strand comprises at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identical to a sequence selected from SEQ ID NOs: 1-70 or SEQ ID NOs:141-210. Alternatively and/or additionally, the polynucleic acidmolecule comprises a sense strand and/or an antisense strand, and theantisense strand comprises at least 80%, at least 85%, at least 9(0%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%identical to a sequence selected from SEQ ID NOs: 71-140 or SEQ ID NOs:211-280.

In certain embodiments, the polynucleotide comprises at least one 2′modified nucleotide, and further the 2′ modified nucleotide comprises2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified nucleotide, or comprises locked nucleic acid (LNA)or ethylene nucleic acid (ENA), or comprises a combination thereof. Incertain embodiments, the at least one modified internucleotide linkagecomprises a phosphorothioate linkage or a phosphorodithioate linkage. Incertain embodiments, the polynucleic acid molecule comprises 3 or more2′ modified nucleotides selected from 2′-O-methyl and2′-deoxy-2′-fluoro. In certain embodiments, the polynucleic acidmolecule comprises a 5′-terminal vinylphosphonate modified nucleotide,such as those described in U.S. Publication No. 2019/0192681.

In certain embodiments, the 2′ modified nucleotide is 2′-O-methylmodified nucleotide, and 2′-O-methyl modified nucleotide is at the5′-end of the sense strand and/or the antisense strand. In someembodiments, the 2′-O-methyl modified nucleotide is a purine nucleotide,or the 2′-O-methyl modified nucleotide is a pyridine nucleotide. Incertain embodiments, the sense and/or antisense strands comprise atleast two, three, four consecutive the 2′-O-methyl modified nucleotidesat the 5′-end.

In certain embodiments, the polynucleic acid molecule conjugatecomprises a linker connecting the target cell binding moiety to thepolynucleic acid moiety. In such embodiments, the linker is C1-C6 alkyllinker, or the linker is a homobifunctional linker or heterobifunctionallinker, and comprises a maleimide group, a dipeptide moiety, a benzoicacid group, or its derivative thereof. Alternatively and/oradditionally, the linker is a cleavable or non-cleavable linker. Incertain embodiments, a ratio between the polynucleic acid moiety and thetarget cell binding moiety is about 1:1, 2:1, 3:1, or 4:1.

In certain embodiments, the polynucleic acid moiety mediates RNAinterference against the human DUX4 and modulates symptoms of muscledystrophy in a subject. In some embodiments, the RNA interferencecomprises reducing expression of the mRNA transcript of DUX4 gene atleast 50%, at least 60%, or at least 70% or more compared to a quantityof the mRNA transcript of DUX4 gene in an untreated cell. Alternativelyand/or additionally, the RNA interference comprises affecting expressionof a marker gene selected from a group comprising or consisting ofMBD3L2, TRIM43, PRAMEF1, ZSCAN4, KHDC1L, and LEUTX in a cell. In someembodiments, the affecting expression of the marker gene is reducingexpression of the marker gene at least 20%, at least 30%, at least 40%,at least 50%, at least 60% or more. In some embodiments, the muscledystrophy is Facioscapulohumeral muscular dystrophy (FSHD).

In certain embodiments, polynucleic acid molecule conjugate comprises amolecule of Formula (I): A-X-B, where A is the antibody or antigenbinding fragment thereof, B is the polynucleic acid molecule thathybridizes to a target sequence of DUX4, X is a bond or a non-polymericlinker, which is conjugated to a cysteine residue of A.

Disclosed herein, in certain embodiments, is a pharmaceuticalcomposition comprising a polynucleic acid molecule conjugate asdescribed herein, and a pharmaceutically acceptable excipient. In someembodiments, the pharmaceutical composition is formulated as ananoparticle formulation. In some embodiments, the pharmaceuticalcomposition is formulated for parenteral, oral, intranasal, buccal,rectal, transdermal, or intravenous, subcutaneous, or intrathecaladministration.

The symptoms of FSHD include affects on skeletal muscles. The skeletalmuscles affected by FSHD include muscles around the eyes and mouth,muscle of the shoulders, muscle of the upper arms, muscle of the lowerlegs, abdominal muscles and hip muscles. In some instances, the symptomsof FSHD also affects vision and hearing. In some instances, the symptomsof FSHD also affect the function of the heart or lungs. In someinstances, the symptoms of FSHD include muscle weakness, muscle atrophy,muscle dystrophy, pain inflammation, contractures, scoliosis, lordosis,hypoventilation, abnormalities of the retina, exposure to keratitis,mild hearing loss, and EMG abnormality. The term muscle atrophy as usedherein refers to a wide range of muscle related effects of FSHD.

Disclosed herein, in certain embodiments, is a method for treatingmuscular dystrophy in a subject in need thereof by providing apolynucleic acid conjugate as described herein, and administering thepolynucleic acid conjugate to the subject in need thereof to treat themuscular dystrophy. The polynucleic acid conjugate reduces a quantity ofthe mRNA transcript of human DUX4. In some embodiments, the polynucleicacid moiety mediates RNA interference against the human DUX4 modulatesmuscle atrophy in a subject. In certain embodiments, the RNAinterference comprises affecting expression of a marker gene selectedfrom a group comprising or consisting of MBD3L2, TRIM43, PRAMEF1,ZSCAN4, KHDCIL, and LEUTX in a cell affected by a muscle dystrophy.Preferably, the muscular dystrophy is Facioscapulohumeral musculardystrophy (FSHD).

Disclosed herein, in certain embodiments, is a use of the polynucleicacid molecule conjugate or a pharmaceutical composition as describedherein for treating in a subject diagnosed with or suspected to haveFacioscapulohumeral muscular dystrophy (FSHD). Also disclosed herein, incertain embodiments, is a use of the polynucleic acid molecule conjugateor the pharmaceutical composition as described herein for manufacturinga medicament for treating in a subject diagnosed with or suspected tohave Facioscapulohumeral muscular dystrophy (FSHD).

Disclosed herein, in certain embodiments, is a kit comprising thepolynucleic acid molecule conjugate or the pharmaceutical composition asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the disclosure are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings below. The patent application file contains atleast one drawing executed in color. Copies of this patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIG. 1 illustrates a diagram of FSHD pathology.

FIG. 2 shows a flowchart diagram of in silico selection of DUX4 siRNA.

FIG. 3 illustrates the location and numbers of selected DUX4 siRNA inthe DUX4 mRNA transcript.

FIG. 4 shows immunofluorescent detection of DUX4 expression inmyonuclei.

FIG. 5 shows bar graphs of siRNA-mediated reduction of DUX4-targetbiomarker gene expressions.

FIGS. 6A-B show graphs of the siRNA-mediated reduction of DUX4-targetbiomarker gene expressions in cultured FSHD primary myotubes and a FSHDcomposite of the siRNA-mediated reduction of DUX4-target biomarker geneexpressions in cultured FSHD primary myotubes

FIG. 7 shows a bar graph of ACTA1 gene expression in myotubes withtreatment of DUX4 siRNA at a concentration of 10 nM.

FIG. 8 shows FSHD composite expression in myotubes with treatment ofDUX4 siRNA at a concentration of 10 nM.

FIG. 9 shows FSHD composite expression in myotubes with treatment ofDUX4 siRNA at a concentration of 0.5 nM.

FIG. 10 shows a flowchart diagram of the selection of DUX4 top 28 siRNAsbased on data from initial screening.

FIGS. 11A-B show FSHD composite expression in two FSHD primary myotubesupon treatment with DUX4 siRNAs at a concentration of 10 nM and 0.5 nM.

FIG. 12 shows a KD correlation analysis identifying DUX4 siRNA effectivein one or more FSHD primary myotubes.

FIG. 13 shows ACTA1 gene expression in patient-derived myotubes withtreatment of DUX4 siRNA at a concentration of 10 nM.

FIG. 14 shows FSHD composite expression in patient-derived myotubes withtreatment of DUX4 siRNA at a concentration of 10 nM.

FIG. 15 shows FSHD composite expression in 6 patient-derived myotubeswith treatment of DUX4 siRNA.

FIGS. 16A-C show graphs of FSHD composite expression in 3patient-derived myotubes with treatment of 14 selected DUX4 siRNAs.

FIGS. 17 A-C show graphs of FSHD composite expression in 3 FSHDpatient-derived myotubes with treatment of 8 selected DUX4 siRNAs.

FIGS. 18 A-B show graphs of FSHD composite expression in cultured FSHDprimary myotubes with treatment of 8 antibody DUX4-siRNA conjugates(DUX4-AOCs) without vinylphosphonate or 8 antibody DUX4-siRNA conjugateswith vinylphosphonate.

FIG. 19 shows graphs of the AOC-mediated in vivo reduction of nuclearlocalized Inc-RNA Malat1 levels in skeletal muscles in mice.

FIG. 20 shows a graph of the sustained SSB-AOC-mediated in vivoreduction of SSB mRNA levels in murine skeletal muscles with a singledose of 3 mg/kg siRNA during an 8-week period.

DETAILED DESCRIPTION OF THE DISCLOSURE

Muscle atrophy is the loss of muscle mass or the progressive weakeningand degeneration of muscles, such as skeletal or voluntary muscles thatcontrols movement, cardiac muscles, and smooth muscles. Variouspathophysiological conditions including disuse, starvation, cancer,diabetes, and renal failure, or treatment with glucocorticoids result inmuscle atrophy and loss of strength. The phenotypical effects of muscleatrophy are induced by various molecular events, including inhibition ofmuscle protein synthesis, enhanced turnover of muscle proteins, abnormalregulation of satellite cells differentiation, and abnormal conversionof muscle fibers types.

FSHD is a rare, progressive and disabling disease for which there are noapproved treatments. FSHD is one of the most common forms of musculardystrophy and affects both sexes equally, with onset typically in teensand young adults. FSHD is characterized by progressive skeletal muscleloss that initially causes weakness in muscles in the face, shoulders,arms and trunk and progresses to weakness in muscles in lowerextremities and the pelvic girdle. Skeletal muscle weakness results insignificant physical limitations, including progressive loss of facialmuscles that can cause an inability to smile or communicate, difficultyusing arms for activities of daily living and difficulty getting out ofbed, with many patients ultimately becoming dependent upon the use of awheelchair for daily mobility activities. The majority of patients withFSHD also report experiencing chronic pain, anxiety and depression.

FSHD is caused by aberrant expression of a gene, DUX4, in skeletalmuscle resulting in the inappropriate presence of DUX4 protein. DUX4itself is a transcription factor that induces the expression of othergenes and it is these inappropriately expressed downstream genes thatresult in the muscle pathology. Normally DUX4-driven gene expression islimited to germline and early stem cell development. In patients withFSHD, the DUX4 protein in skeletal muscle regulates other gene products,some of which are toxic to the muscle. Evidence of aberrant DUX4-drivengene expression is the major molecular signature that distinguishesmuscle tissue affected by FSHD from healthy muscle. The result ofaberrant DUX4 expression in FSHD is death of muscle and its replacementby fat, resulting in skeletal muscle weakness and progressivedisability. Data suggest that reducing expression of the DUX4 gene andits downstream transcriptional program could provide a disease-modifyingtherapeutic approach for the treatment of FSHD at its root cause.

There are two ways the DUX4 gene can be unsilenced, or de-repressed. InFSHD1, which comprises approximately 95% of FSHD patients, there aremutations that lead to the shortening of an array of DNA in a regionnear the end of the long arm of chromosome 4, known as D4Z4, which hasrepeats in the subtelomeric region of the chromosome. The D4Z4 region isabnormally shortened and contains between 1-10 repeats instead of thenormal 11 to 100 repeats. This contraction causes hypomethylation of theD4Z4 region and de-repression of DUX4. Patients with FSHD2 do not have ameaningful D4Z4 repeat contraction, but have mutations in a regulatorygene, known as the SMCHD1 gene, that normally contributes to therepression of the DUX4 gene via DNA methylation. When that repression islost due to the mutations of the SMCHD1 gene leading to thehypomethylation of the D4Z4 region, DUX4 is inappropriately expressed,inducing the disease state. FIG. 1 shows an illustrative diagram of FSHDpathology.

Nucleic acid (e.g., RNAi) therapy is a targeted therapy with highselectivity and specificity. However, in some instances, nucleic acidtherapy is also hindered by poor intracellular uptake, limited bloodstability and non-specific immune stimulation. To address these issues,various modifications of the nucleic acid composition are explored, suchas for example, novel linkers for better stabilizing and/or lowertoxicity, optimization of binding moiety for increased targetspecificity and/or target delivery, and nucleic acid polymermodifications for increased stability and/or reduced off-target effect.

In some embodiments, the arrangement or order of the differentcomponents that make-up the nucleic acid composition further effectsintracellular uptake, stability, toxicity, efficacy, and/or non-specificimmune stimulation. For example, if the nucleic acid component includesa binding moiety, a polymer, and a polynucleic acid molecule (orpolynucleotide), the order or arrangement of the binding moiety, thepolymer, and/or the polynucleic acid molecule (or polynucleotide) (e.g.,binding moiety-polynucleic acid molecule-polymer, bindingmoiety-polymer-polynucleic acid molecule, or polymer-bindingmoiety-polynucleic acid molecule) further effects intracellular uptake,stability, toxicity, efficacy, and/or non-specific immune stimulation.

In some embodiments, described herein include polynucleic acid moleculesand polynucleic acid molecule conjugates for the treatment ofFacioscapulohumeral Muscular Dystrophy (FSHD) especially muscledystrophy and/or muscle atrophy associated therewith. In some instances,the polynucleic acid molecule conjugates described herein enhanceintracellular uptake, stability, and/or efficacy. In some cases, thepolynucleic acid molecule conjugates comprise an antibody or antigenbinding fragment thereof conjugated to a polynucleic acid molecule. Insome cases, the polynucleic acid molecules that hybridize to targetsequences of DUX4, preferably human DUX4.

Additional embodiments described herein include methods of treatingFSHD, comprising administering to a subject a polynucleic acid moleculeor a polynucleic acid molecule conjugate described herein.

Polynucleic Acid Molecules

In certain embodiments, a polynucleic acid molecule hybridizes to atarget sequence of Double homeobox 4 (DUX4) gene. In some instances, apolynucleic acid molecule described herein hybridizes to a targetsequence of human DUX4 gene (DUX4) and reduces DUX4 mRNA in musclecells.

In some embodiments, the polynucleic acid molecule comprises a sequencehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQID NOs: 1-70. In some embodiments, the polynucleic acid moleculecomprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to asequence selected from SEQ ID NOs: 141-210. In some embodiments, thepolynucleic acid molecule comprises a sequence having at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to a sequence selected from SEQ ID NOs: 71-140. Insome embodiments, the polynucleic acid molecule comprises a sequencehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQID NOs: 211-280.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide and a second polynucleotide. In some instances, the firstpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence selected from SEQ ID NOs: 1-70. In some cases,the second polynucleotide comprises a sequence having at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to a sequence selected from SEQ ID NOs: 71-140. Insome cases, the polynucleic acid molecule comprises a firstpolynucleotide and a second polynucleotide. In some instances, the firstpolynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence selected from SEQ ID NOs: 141-210. In some cases,the second polynucleotide comprises a sequence having at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100/sequence identity to a sequence selected from SEQ ID NOSs: 211-280.

In some embodiments, the polynucleic acid molecule comprises a sensestrand (e.g., a passenger strand) and an antisense strand (e.g., a guidestrand). In some instances, the sense strand (e.g., the passengerstrand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto a sequence selected from SEQ ID NOs: 1-70. In some instances, theantisense strand (e.g., the guide strand) comprises a sequence having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to a sequence selected from SEQ ID NOs:71-140. In some embodiments, the polynucleic acid molecule comprises asense strand (e.g., a passenger strand) and an antisense strand (e.g., aguide strand). In some instances, the sense strand (e.g., the passengerstrand) comprises a sequence having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto a sequence selected from SEQ ID NOs: 141-210. In some instances, theantisense strand (e.g., the guide strand) comprises a sequence having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to a sequence selected from SEQ ID NOs:211-280.

In some instances, the sense strand comprises a sequence having at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to a sequence selected from SEQ ID NOs: 1, 2, 3,6, 14, 36, 52, 56, 61, 62, 63, 65, 66. In some instances, the antisensestrand comprises a sequence having at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identityto a sequence selected from 71, 72, 73, 76, 84, 106, 122, 127, 131, 132,133, 135, 136. In some instances, the siRNA comprises sense strand andantisense strand as presented in Table 11.

In some instances, the sense strand comprises a sequence having at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to a sequence selected from SEQ ID NOs: 141, 142,143, 146, 176, 192, 196, 201, 202, 203, 205, 206. In some instances, theantisense strand comprises a sequence having at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to a sequence selected from SEQ ID NOs: 211, 212, 213, 216,246, 262, 266, 271, 272, 273, 275, 276. In some instances, the siRNAcomprises sense strand and antisense strand as presented in Table 12.

In some embodiments, the polynucleic acid molecule described hereincomprises RNA or DNA. In some cases, the polynucleic acid moleculecomprises RNA. In some instances, RNA comprises short interfering RNA(siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-strandedRNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), or heterogeneousnuclear RNA (hnRNA). In some instances, RNA comprises shRNA. In someinstances, RNA comprises miRNA. In some instances, RNA comprises dsRNA.In some instances. RNA comprises tRNA. In some instances, RNA comprisesrRNA. In some instances, RNA comprises hnRNA. In some instances, theoligonucleotide is a phosphorodiamidate morpholino oligomers (PMO),which are short single-stranded oligonucleotide analogs that are builtupon a backbone of morpholine rings connected by phosphorodianidatelinkages. In some instances, the RNA comprises siRNA. In some instances,the polynucleic acid molecule comprises siRNA.

In some embodiments, the polynucleic acid molecule is from about 8 toabout 50 nucleotides in length. In some embodiments, the polynucleicacid molecule is from about 10 to about 50 nucleotides in length. Insome instances, the polynucleic acid molecule is from about 10 to about30, from about 15 to about 30, from about 18 to about 25, form about 18to about 24, from about 19 to about 23, or from about 20 to about 22nucleotides in length.

In some embodiments, the polynucleic acid molecule is about 50nucleotides in length. In some instances, the polynucleic acid moleculeis about 45 nucleotides in length. In some instances, the polynucleicacid molecule is about 40 nucleotides in length. In some instances, thepolynucleic acid molecule is about 35 nucleotides in length. In someinstances, the polynucleic acid molecule is about 30 nucleotides inlength. In some instances, the polynucleic acid molecule is about 25nucleotides in length. In some instances, the polynucleic acid moleculeis about 20 nucleotides in length. In some instances, the polynucleicacid molecule is about 19 nucleotides in length. In some instances, thepolynucleic acid molecule is about 18 nucleotides in length. In someinstances, the polynucleic acid molecule is about 17 nucleotides inlength. In some instances, the polynucleic acid molecule is about 16nucleotides in length. In some instances, the polynucleic acid moleculeis about 15 nucleotides in length. In some instances, the polynucleicacid molecule is about 14 nucleotides in length. In some instances, thepolynucleic acid molecule is about 13 nucleotides in length. In someinstances, the polynucleic acid molecule is about 12 nucleotides inlength. In some instances, the polynucleic acid molecule is about 11nucleotides in length. In some instances, the polynucleic acid moleculeis about 10 nucleotides in length. In some instances, the polynucleicacid molecule is about 8 nucleotides in length. In some instances, thepolynucleic acid molecule is between about 8 and about 50 nucleotides inlength. In some instances, the polynucleic acid molecule is betweenabout 10 and about 50 nucleotides in length. In some instances, thepolynucleic acid molecule is between about 10 and about 45 nucleotidesin length. In some instances, the polynucleic acid molecule is betweenabout 10 and about 40 nucleotides in length. In some instances, thepolynucleic acid molecule is between about 10 and about 35 nucleotidesin length. In some instances, the polynucleic acid molecule is betweenabout 10 and about 30 nucleotides in length. In some instances, thepolynucleic acid molecule is between about 10 and about 25 nucleotidesin length. In some instances, the polynucleic acid molecule is betweenabout 10 and about 20 nucleotides in length. In some instances, thepolynucleic acid molecule is between about 15 and about 25 nucleotidesin length. In some instances, the polynucleic acid molecule is betweenabout 15 and about 30 nucleotides in length. In some instances, thepolynucleic acid molecule is between about 12 and about 30 nucleotidesin length.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide. In some instances, the polynucleic acid moleculecomprises a second polynucleotide. In some instances, the polynucleicacid molecule comprises a first polynucleotide and a secondpolynucleotide. In some instances, the first polynucleotide is a sensestrand or passenger strand. In some instances, the second polynucleotideis an antisense strand or guide strand.

In some embodiments, the polynucleic acid molecule is a firstpolynucleotide. In some embodiments, the first polynucleotide is fromabout 8 to about 50 nucleotides in length. In some embodiments, thefirst polynucleotide is from about 10 to about 50 nucleotides in length.In some instances, the first polynucleotide is from about 10 to about30, from about 15 to about 30, from about 18 to about 25, form about 18to about 24, from about 19 to about 23, or from about 20 to about 22nucleotides in length.

In some instances, the first polynucleotide is about 50 nucleotides inlength. In some instances, the first polynucleotide is about 45nucleotides in length. In some instances, the first polynucleotide isabout 40 nucleotides in length. In some instances, the firstpolynucleotide is about 35 nucleotides in length. In some instances, thefirst polynucleotide is about 30 nucleotides in length. In someinstances, the first polynucleotide is about 25 nucleotides in length.In some instances, the first polynucleotide is about 20 nucleotides inlength. In some instances, the first polynucleotide is about 19nucleotides in length. In some instances, the first polynucleotide isabout 18 nucleotides in length. In some instances, the firstpolynucleotide is about 17 nucleotides in length. In some instances, thefirst polynucleotide is about 16 nucleotides in length. In someinstances, the first polynucleotide is about 15 nucleotides in length.In some instances, the first polynucleotide is about 14 nucleotides inlength. In some instances, the first polynucleotide is about 13nucleotides in length. In some instances, the first polynucleotide isabout 12 nucleotides in length. In some instances, the firstpolynucleotide is about 11 nucleotides in length. In some instances, thefirst polynucleotide is about 10 nucleotides in length. In someinstances, the first polynucleotide is about 8 nucleotides in length. Insome instances, the first polynucleotide is between about 8 and about 50nucleotides in length. In some instances, the first polynucleotide isbetween about 10 and about 50 nucleotides in length. In some instances,the first polynucleotide is between about 10 and about 45 nucleotides inlength. In some instances, the first polynucleotide is between about 10and about 40 nucleotides in length. In some instances, the firstpolynucleotide is between about 10 and about 35 nucleotides in length.In some instances, the first polynucleotide is between about 10 andabout 30 nucleotides in length. In some instances, the firstpolynucleotide is between about 10 and about 25 nucleotides in length.In some instances, the first polynucleotide is between about 10 andabout 20 nucleotides in length. In some instances, the firstpolynucleotide is between about 15 and about 25 nucleotides in length.In some instances, the first polynucleotide is between about 15 andabout 30 nucleotides in length. In some instances, the firstpolynucleotide is between about 12 and about 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule is a secondpolynucleotide. In some embodiments, the second polynucleotide is fromabout 8 to about 50 nucleotides in length. In some embodiments, thesecond polynucleotide is from about 10 to about 50 nucleotides inlength. In some instances, the second polynucleotide is from about 10 toabout 30, from about 15 to about 30, from about 18 to about 25, formabout 18 to about 24, from about 19 to about 23, or from about 20 toabout 22 nucleotides in length.

In some instances, the second polynucleotide is about 50 nucleotides inlength. In some instances, the second polynucleotide is about 45nucleotides in length. In some instances, the second polynucleotide isabout 40 nucleotides in length. In some instances, the secondpolynucleotide is about 35 nucleotides in length. In some instances, thesecond polynucleotide is about 30 nucleotides in length. In someinstances, the second polynucleotide is about 25 nucleotides in length.In some instances, the second polynucleotide is about 20 nucleotides inlength. In some instances, the second polynucleotide is about 19nucleotides in length. In some instances, the second polynucleotide isabout 18 nucleotides in length. In some instances, the secondpolynucleotide is about 17 nucleotides in length. In some instances, thesecond polynucleotide is about 16 nucleotides in length. In someinstances, the second polynucleotide is about 15 nucleotides in length.In some instances, the second polynucleotide is about 14 nucleotides inlength. In some instances, the second polynucleotide is about 13nucleotides in length. In some instances, the second polynucleotide isabout 12 nucleotides in length. In some instances, the secondpolynucleotide is about 11 nucleotides in length. In some instances, thesecond polynucleotide is about 10 nucleotides in length. In someinstances, the second polynucleotide is about 8 nucleotides in length.In some instances, the second polynucleotide is between about 8 andabout 50 nucleotides in length. In some instances, the secondpolynucleotide is between about 10 and about 50 nucleotides in length.In some instances, the second polynucleotide is between about 10 andabout 45 nucleotides in length. In some instances, the secondpolynucleotide is between about 10 and about 40 nucleotides in length.In some instances, the second polynucleotide is between about 10 andabout 35 nucleotides in length. In some instances, the secondpolynucleotide is between about 10 and about 30 nucleotides in length.In some instances, the second polynucleotide is between about 10 andabout 25 nucleotides in length. In some instances, the secondpolynucleotide is between about 10 and about 20 nucleotides in length.In some instances, the second polynucleotide is between about 15 andabout 25 nucleotides in length. In some instances, the secondpolynucleotide is between about 15 and about 30 nucleotides in length.In some instances, the second polynucleotide is between about 12 andabout 30 nucleotides in length.

In some embodiments, the polynucleic acid molecule comprises a firstpolynucleotide and a second polynucleotide. In some instances, thepolynucleic acid molecule further comprises a blunt terminus, anoverhang, or a combination thereof. In some instances, the bluntterminus is a 5′ blunt terminus, a 3′ blunt terminus, or both. In somecases, the overhang is a 5′ overhang, 3′ overhang, or both. In somecases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-basepairing nucleotides. In some cases, the overhang comprises 1, 2, 3, 4,5, or 6 non-base pairing nucleotides. In some cases, the overhangcomprises 1, 2, 3, or 4 non-base pairing nucleotides. In some cases, theoverhang comprises 1 non-base pairing nucleotide. In some cases, theoverhang comprises 2 non-base pairing nucleotides. In some cases, theoverhang comprises 3 non-base pairing nucleotides. In some cases, theoverhang comprises 4 non-base pairing nucleotides. In some embodiments,the polynucleic acid molecule comprises a sense strand and an antisensestrand, and the antisense strand includes two non-base pairingnucleotides as an overhang at the 3′-end while the sense strand has nooverhang. Optionally, in such embodiments, the non-base pairingnucleotides have a sequence of TT, dTdT, or UU. In some embodiments, thepolynucleic acid molecule comprises a sense strand and an antisensestrand, and the sense strand has one or more nucleotides at the 5′-endthat are complementary to the antisense sequence.

In some embodiments, the sequence of the polynucleic acid molecule is atleast 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%,or 99.5% complementary to a target sequence of DUX4. In someembodiments, the target sequence of DUX4 is a nucleic acid sequence ofabout 10-50 base pair length, about 15-50 base pair length, 15-40 basepair length, 15-30 base pair length, or 15-25 base pair length sequencesin DUX4, in which the first nucleotide of the target sequence starts atany nucleotide in DUX4 mRNA transcript in the coding region, or in the5′ or 3′-untranslated region (UTR). For example, the first nucleotide ofthe target sequence can be selected so that it starts at the nucleicacid location (nal, number starting from the 5′-end of the full lengthof DUX mRNA, e.g., the 5-end first nucleotide is nal.1) 1, nal 2, nal 3,nal 4, nal 5, nal 6, nal 7, nal 8, nal 9, nal 10, nal 11, nal 12, nal13, nal 14, nal 15, nal 15, nal 16, nal 17, or any other nucleic acidlocation in the coding or noncoding regions (5′ or 3′-untraslatedregion) of DUX mRNA. In some embodiments, the first nucleotide of thetarget sequence can be selected so that it starts at a location within,or between, nal 10-nal 15, nal 10-nal 20, nal 50-nal 60, nal 55-nal 65,nal 75-nal 85, nal 95-nal 105, nal 135-nal 145, nal 155-nal 165, nal225-nal 235, nal 265-nal 275, nal 275-nal 285, nal 285-nal 295, nal325-nal 335, nal 335-nal 345, nal 385-nal 395, nal 515-nal 525, nal665-nal 675, nal 675-nal 685, nal 695-nal 705, nal 705-nal 715, nal875-nal 885, nal 885-nal 895, nal 895-nal 905, nal 1035-nal 1045, nal1045-nal 1055, nal 1125-nal 1135, nal 1135-nal 1145, nal 1145-nal 1155,nal 1155-nal 1165, nal 1125-nal 1135, nal 1155-nal 1165, nal 1225-nal1235, nal 1235-nal 1245, nal 1275-nal 1285, nal 1285-nal 1295, nal1305-nal 1315, nal 1125-nal 1135, nal 1155-nal 1165, nal 1225-nal 1235,nal 1235-nal 1245, nal 1275-nal 1285, nal 1285-nal 1295, nal 1305-nal1315, nal 1315-nal 1325, nal 1335-nal 1345, nal 1345-nal 1355, nal1525-nal 1535, nal 1535-nal 1545, nal 1605-nal 1615, nal 1615-c.1625,nal 1625-nal 1635.

In some embodiments, the sequence of the polynucleic acid molecule is atleast 50% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least60% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least70% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least80% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least90% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least95% complementary to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule is at least99% complementary to a target sequence described herein. In someinstances, the sequence of the polynucleic acid molecule is 100%complementary to a target sequence described herein.

In some embodiments, the sequence of the polynucleic acid molecule has 5or less mismatches to a target sequence described herein. In someembodiments, the sequence of the polynucleic acid molecule has 4 or lessmismatches to a target sequence described herein. In some instances, thesequence of the polynucleic acid molecule has 3 or less mismatches to atarget sequence described herein. In some cases, the sequence of thepolynucleic acid molecule has 2 or less mismatches to a target sequencedescribed herein. In some cases, the sequence of the polynucleic acidmolecule has 1 or less mismatches to a target sequence described herein.

In some embodiments, a group of polynucleic acid molecules among all thepolynucleic acid molecules potentially binds to the target sequence ofDUX4 are selected to generate a polynucleic acid molecule library. Incertain embodiments, such selection process is conducted in silico viaone or more steps of eliminating less desirable polynucleic acidmolecules from candidates. For example, in some embodiments, theselection process comprises an elimination step of one or morepolynucleic acid molecule that has single nucleotide polymorphism (SNP)and/or MEF<−5. Alternatively and/or additionally, in some embodiments,the selection process comprises an elimination step of one or morepolynucleic acid molecule with 0 and 1 mismatch (MM) in the humantranscriptome (such that only hits allowed are DUX, DUX5, and DBET).Alternatively and/or additionally, in some embodiments, the selectionprocess comprises an elimination step of one or more polynucleic acidmolecule with 0 MM in the human intragenic regions (such that only hitsallowed are DUX1. DUX5 and DBET pseudogenes). Alternatively and/oradditionally, in some embodiments, the selection process comprises anelimination step of one or more polynucleic acid molecule with a MM toDUX4 human sequence used in FLExDUX4 FSHD mouse model. Alternativelyand/or additionally, in some embodiments, the selection processcomprises an elimination step of one or more polynucleic acid moleculepredicted viability<60. Alternatively and/or additionally, suchselection process comprises carrying forward one or more polynucleicacid molecule predicted viability>60. Alternatively and/or additionally,in some embodiments, the selection process comprises an elimination stepof one or more polynucleic acid molecule with a match to a seed regionof known miRNAs 1-1000. Alternatively and/or additionally, in someembodiments, the selection process comprises an elimination step of oneor more polynucleic acid molecule with % GC content 75 and above.Alternatively and/or additionally, in some embodiments, the selectionprocess comprises a selection step of 8 or less predicted off-targethits with 2 MM. In some embodiments, for the region 295-1132 (nal295-1132), 12 or less predicted off-target hits with 2 MM is allowed.

In some embodiments, selection process is conducted in silico via one ormore consecutive steps of eliminating less desirable polynucleic acidmolecules from candidates. For example, in some embodiments, selectionprocess begins with collecting candidate polynucleic acid molecules togenerate a library. From the library, the first eliminating stepcomprises eliminating one or more polynucleic acid molecule that hassingle nucleotide polymorphism (SNP) and/or MEF<−5. Then, the secondeliminating step comprises eliminating one or more polynucleic acidmolecule with 0 and 1 MM in the human transcriptome (such that only hitsallowed are DUX, DUX5, and DBET). Then, the third eliminating stepcomprises eliminating one or more polynucleic acid molecule with 0 MM inthe human intragenic regions (such that only hits allowed are DUX1, DUX5and DBET pseudogenes). Then, the next eliminating step compriseseliminating one or more polynucleic acid molecule with a MM to DUX4human sequence used in FLExDUX4 FSHD mouse model. Then, the next step iscarrying forward only or one or more polynucleic acid molecule withpredicted viability>60. Next, the eliminating step comprises eliminatingone or more polynucleic acid molecule with a match to a seed region ofknown miRNAs 1-1000. Then, the eliminating step continues witheliminating one or more polynucleic acid molecule with % GC content 75and above. Then, the final selection process comprises with 8 or lesspredicted off-target hits with 2 MM, except for the region 295-1132, forwhich up to 12 hits are allowed.

In some embodiments, the specificity of the polynucleic acid moleculethat hybridizes to a target sequence described herein is a 95%, 98%,99%, 99.5% or 100% sequence complementarity of the polynucleic acidmolecule to a target sequence. In some instances, the hybridization is ahigh stringent hybridization condition.

In some embodiments, the polynucleic acid molecule has reducedoff-target effect. In some instances, “off-target” or “off-targeteffects” refer to any instance in which a polynucleic acid polymerdirected against a given target causes an unintended effect byinteracting either directly or indirectly with another mRNA sequence, aDNA sequence or a cellular protein or other moiety. In some instances,an “off-target effect” occurs when there is a simultaneous degradationof other transcripts due to partial homology or complementarity betweenthat other transcript and the sense and/or antisense strand of thepolynucleic acid molecule.

In some embodiments, the polynucleic acid molecule comprises natural orsynthetic or artificial nucleotide analogues or bases. In some cases,the polynucleic acid molecule comprises combinations of DNA, RNA and/ornucleotide analogues. In some instances, the synthetic or artificialnucleotide analogues or bases comprise modifications at one or more ofribose moiety, phosphate moiety, nucleoside moiety, or a combinationthereof.

In some embodiments, nucleotide analogues or artificial nucleotide basecomprise a nucleic acid with a modification at a 2′ hydroxyl group ofthe ribose moiety. In some instances, the modification includes an H,OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety.Exemplary alkyl moiety includes, but is not limited to, halogens,sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, ortertiary), amides, ethers, esters, alcohols and oxygen. In someinstances, the alkyl moiety further comprises a modification. In someinstances, the modification comprises an azo group, a keto group, analdehyde group, a carboxyl group, a nitro group, a nitroso, group, anitrile group, a heterocycle (e.g., imidazole, hydrazino orhydroxylamino) group, an isocyanate or cyanate group, or a sulfurcontaining group (e.g., sulfoxide, sulfone, sulfide, and disulfide). Insome instances, the alkyl moiety further comprises a heterosubstitution. In some instances, the carbon of the heterocyclic group issubstituted by a nitrogen, oxygen or sulfur. In some instances, theheterocyclic substitution includes but is not limited to, morpholino,imidazole, and pyrrolidino.

In some instances, the modification at the 2′ hydroxyl group is a2′-O-methyl modification or a 2′-O-methoxyethyl (2′-O-MOE) modification.In some cases, the 2′-O-methyl modification adds a methyl group to the2′ hydroxyl group of the ribose moiety whereas the 2′O-methoxyethylmodification adds a methoxyethyl group to the 2′ hydroxyl group of theribose moiety. Exemplary chemical structures of a 2′-O-methylmodification of an adenosine molecule and 2′O-methoxyethyl modificationof an uridine are illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a2′-O-aminopropyl modification in which an extended amine groupcomprising a propyl linker binds the amine group to the 2′ oxygen. Insome instances, this modification neutralizes the phosphate derivedoverall negative charge of the oligonucleotide molecule by introducingone positive charge from the amine group per sugar and thereby improvescellular uptake properties due to its zwitterionic properties. Anexemplary chemical structure of a 2′-O-aminopropyl nucleosidephosphoramidite is illustrated below.

In some instances, the modification at the 2′ hydroxyl group is a lockedor bridged ribose modification (e.g., locked nucleic acid or LNA) inwhich the oxygen molecule bound at the 2′ carbon is linked to the 4′carbon by a methylene group, thus forming a2′-C,4′-C-oxy-methylene-linked bicyclic ribonucleotide monomer.Exemplary representations of the chemical structure of LNA areillustrated below. The representation shown to the left highlights thechemical connectivities of an LNA monomer. The representation shown tothe right highlights the locked 3′-endo (³E) conformation of thefuranose ring of an LNA monomer.

In some instances, the modification at the 2′ hydroxyl group comprisesethylene nucleic acids (ENA) such as for example 2′-4′-ethylene-bridgednucleic acid, which locks the sugar conformation into a C₃′-endo sugarpuckering conformation. ENA are part of the bridged nucleic acids classof modified nucleic acids that also comprises LNA. Exemplary chemicalstructures of the ENA and bridged nucleic acids are illustrated below.

In some embodiments, additional modifications at the 2′ hydroxyl groupinclude 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA).

In some embodiments, nucleotide analogues comprise modified bases suchas, but not limited to, 5-propynyluridine, 5-propynylcytidine,6-methyladenine, 6-methylguanine, N, N, -dimethyladenine,2-propyladenine, 2-propylguanine, 2-aminoadenine, 1-methylinosine,3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotideshaving a modification at the 5 position, 5-(2-amino) propyl uridine,5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine,2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine,7-methylguanosine, 2, 2-dimethylguanosine, 5-methylaminoethyluridine,5-methyloxyuridine, deazanucleotides such as 7-deaza-adenosine,6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2-thiouridine,other thio bases such as 2-thiouridine and 4-thiouridine and2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine,naphthyl and substituted naphthyl groups, any O- and N-alkylated purinesand pyrimidines such as N6-methyladenosine,5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one,pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or2,4, 6-trimethoxy benzene, modified cytosines that act as G-clampnucleotides, 8-substituted adenines and guanines, 5-substituted uracilsand thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides,carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylatednucleotides. Modified nucleotides also include those nucleotides thatare modified with respect to the sugar moiety, as well as nucleotideshaving sugars or analogs thereof that are not ribosyl. For example, thesugar moieties, in some cases are or be based on, mannoses, arabinoses,glucopyranoses, galactopyranoses, 4′-thioribose, and other sugars,heterocycles, or carbocycles. The term nucleotide also includes what areknown in the art as universal bases. By way of example, universal basesinclude but are not limited to 3-nitropyrrole, 5-nitroindole, ornebularine.

In some embodiments, nucleotide analogues further comprise morpholinos,peptide nucleic acids (PNAs), methylphosphonate nucleotides,thiolphosphonate nucleotides, 2′-fluoro N3-P5′-phosphoramidites, 1′,5′-anhydrohexitol nucleic acids (HNAs), or a combination thereof.Morpholino or phosphorodiamidate morpholino oligo (PMO) comprisessynthetic molecules whose structure mimics natural nucleic acidstructure by deviates from the normal sugar and phosphate structures. Insome instances, the five member ribose ring is substituted with a sixmember morpholino ring containing four carbons, one nitrogen and oneoxygen. In some cases, the ribose monomers are linked by aphosphordianidate group instead of a phosphate group. In such cases, thebackbone alterations remove all positive and negative charges makingmorpholinos neutral molecules capable of crossing cellular membraneswithout the aid of cellular delivery agents such as those used bycharged oligonucleotides.

In some embodiments, peptide nucleic acid (PNA) does not contain sugarring or phosphate linkage and the bases are attached and appropriatelyspaced by oligoglycine-like molecules, therefore, eliminating a backbonecharge.

In some embodiments, one or more modifications optionally occur at theinternucleotide linkage. In some instances, modified internucleotidelinkage include, but is not limited to, phosphorothioates,phosphorodithioates, methylphosphonates, 5′-alkylenephosphonates,5′-methylphosphonate, 3′-alkylene phosphonates, borontrifluoridates,borano phosphate esters and selenophosphates of 3′-5′ linkage or 2′-5′linkage, phosphotriesters, thionoalkylphosphotriesters, hydrogenphosphonate linkages, alkyl phosphonates, alkylphosphonothioates,arylphosphonothioates, phosphoroselenoates, phosphorodiselenoates,phosphinates, phosphoramidates, 3′-alkylphosphoramidates,aminoalkylphosphoramidates, thionophosphoramidates,phosphoropiperazidates, phosphoroanilothioates, phosphoroanilidates,ketones, sulfones, sulfonamides, carbonates, carbamates,methylenehydrazos, methylenedimethylhydrazos, formacetals,thioformacetals, oximes, methyleneiminos, methylenemethyliminos,thioamidates, linkages with riboacetyl groups, aminoethyl glycine, silylor siloxane linkages, alkyl or cycloalkyl linkages with or withoutheteroatoms of, for example, 1 to 10 carbons that are saturated orunsaturated and/or substituted and/or contain heteroatoms, linkages withmorpholino structures, amides, polyamides wherein the bases are attachedto the aza nitrogens of the backbone directly or indirectly, andcombinations thereof. Phosphorothioate antisense oligonucleotides (PSASO) are antisense oligonucleotides comprising a phosphorothioatelinkage. An exemplary PS ASO is illustrated below.

In some instances, the modification is a methyl or thiol modificationsuch as methylphosphonate or thiolphosphonate modification. Exemplarythiolphosphonate nucleotide (left) and methylphosphonate nucleotide(right) are illustrated below.

In some instances, a modified nucleotide includes, but is not limitedto, 2′-fluoro N3-P5′-phosphoramidites illustrated as:

In some instances, a modified nucleotide includes, but is not limitedto, hexitol nucleic acid (or 1′, 5′-anhydrohexitol nucleic acids (1HNA))illustrated as:

In some embodiments, one or more modifications further optionallyinclude modifications of the ribose moiety, phosphate backbone and thenucleoside, or modifications of the nucleotide analogues at the 3′ orthe 5′ terminus. For example, the 3′ terminus optionally include a 3′cationic group, or by inverting the nucleoside at the 3′-terminus with a3′-3′ linkage. In another alternative, the 3′-terminus is optionallyconjugated with an aminoalkyl group, e.g. a 3′ C5-aminoalkyl dT. In anadditional alternative, the 3′-terminus is optionally conjugated with anabasic site, e.g., with an apurinic or apyrimidinic site. In someinstances, the 5″-terminus is conjugated with an aminoalkyl group. e.g.a 5′-O-alkylamino substituent. In some cases, the 5′-terminus isconjugated with an abasic site. e.g., with an apurinic or apyrimidinicsite.

In some embodiments, the polynucleic acid molecule comprises one or moreof the artificial nucleotide analogues described herein. In someinstances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of theartificial nucleotide analogues described herein. In some embodiments,the artificial nucleotide analogues include 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or a combination thereof. In some instances,the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificialnucleotide analogues selected from 2′-O-methyl, 2′-O-methoxyethyl(2′-O-MOE), 2-O-aminopropyl, 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA,PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonatenucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combinationthereof. In some instances, the polynucleic acid molecule comprises 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, ormore of 2′-O-methyl modified nucleotides. In some instances, thepolynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2′-O-methoxyethyl(2′-O-MOE) modified nucleotides. In some instances, the polynucleic acidmolecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 20, 25, or more of thiolphosphonate nucleotides.

In some instances, the polynucleic acid molecule comprises at least oneof: from about 5% to about 100% modification, from about 10% to about100% modification, from about 20% to about 100% modification, from about30% to about 100% modification, from about 40% to about 100%modification, from about 50% to about 100% modification, from about 60%to about 100% modification, from about 70% to about 100% modification,from about 80% to about 100% modification, and from about 90% to about100% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 90% modification, from about 20% to about 90%modification, from about 30% to about 90% modification, from about 40%to about 90% modification, from about 50% to about 90% modification,from about 60% to about 90% modification, from about 70% to about 90%modification, and from about 80% to about 100% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 80% modification, from about 20% to about 80%modification, from about 30% to about 80% modification, from about 40%to about 80% modification, from about 50% to about 80% modification,from about 60% to about 80% modification, and from about 70% to about80% modification.

In some instances, the polynucleic acid molecule comprises at least oneof: from about 10% to about 70% modification, from about 20% to about70% modification, from about 30% to about 70% modification, from about40% to about 70% modification, from about 50% to about 70% modification,and from about 60% to about 70% modification.

In some instances, the polynucleic acid molecule comprises at least oneof: from about 10% to about 60% modification, from about 20% to about60% modification, from about 30% to about 60% modification, from about40% to about 60% modification, and from about 50% to about 60%modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 50% modification, from about 20% to about 50%modification, from about 30% to about 50% modification, and from about40% to about 50% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 40% modification, from about 20% to about 40%modification, and from about 30% to about 40% modification.

In some cases, the polynucleic acid molecule comprises at least one of:from about 10% to about 30% modification, and from about 20% to about30% modification.

In some cases, the polynucleic acid molecule comprises from about 10% toabout 20% modification.

In some cases, the polynucleic acid molecule comprises from about 15% toabout 90%, from about 20% to about 80%, from about 30% to about 70%, orfrom about 40% to about 60% modifications.

In additional cases, the polynucleic acid molecule comprises at leastabout 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%modification.

In some embodiments, the polynucleic acid molecule comprises at leastabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22 ormore modifications.

In some instances, the polynucleic acid molecule comprises at leastabout 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8,about 9, about 10, about 11, about 12, about 13, about 14, about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22 ormore modified nucleotides.

In some instances, from about 5 to about 100% of the polynucleic acidmolecule comprise the artificial nucleotide analogues described herein.In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of thepolynucleic acid molecule comprise the artificial nucleotide analoguesdescribed herein. In some instances, about 5% of the polynucleic acidmolecule comprises the artificial nucleotide analogues described herein.In some instances, about 10% of the polynucleic acid molecule comprisesthe artificial nucleotide analogues described herein. In some instances,about 15% of the polynucleic acid molecule comprises the artificialnucleotide analogues described herein. In some instances, about 20% ofthe polynucleic acid molecule comprises the artificial nucleotideanalogues described herein. In some instances, about 25% of thepolynucleic acid molecule comprises the artificial nucleotide analoguesdescribed herein. In some instances, about 30% of the polynucleic acidmolecule comprises the artificial nucleotide analogues described herein.In some instances, about 35% of the polynucleic acid molecule comprisesthe artificial nucleotide analogues described herein. In some instances,about 40% of the polynucleic acid molecule comprises the artificialnucleotide analogues described herein. In some instances, about 45% ofthe polynucleic acid molecule comprises the artificial nucleotideanalogues described herein. In some instances, about 50% of thepolynucleic acid molecule comprises the artificial nucleotide analoguesdescribed herein. In some instances, about 55% of the polynucleic acidmolecule comprises the artificial nucleotide analogues described herein.In some instances, about 60% of the polynucleic acid molecule comprisesthe artificial nucleotide analogues described herein. In some instances,about 65% of the polynucleic acid molecule comprises the artificialnucleotide analogues described herein. In some instances, about 70% ofthe polynucleic acid molecule comprises the artificial nucleotideanalogues described herein. In some instances, about 75% of thepolynucleic acid molecule comprises the artificial nucleotide analoguesdescribed herein. In some instances, about 80% of the polynucleic acidmolecule comprises the artificial nucleotide analogues described herein.In some instances, about 85% of the polynucleic acid molecule comprisesthe artificial nucleotide analogues described herein. In some instances,about 90% of the polynucleic acid molecule comprises the artificialnucleotide analogues described herein. In some instances, about 95% ofthe polynucleic acid molecule comprises the artificial nucleotideanalogues described herein. In some instances, about 96% of thepolynucleic acid molecule comprises the artificial nucleotide analoguesdescribed herein. In some instances, about 97% of the polynucleic acidmolecule comprises the artificial nucleotide analogues described herein.In some instances, about 98% of the polynucleic acid molecule comprisesthe artificial nucleotide analogues described herein. In some instances,about 99% of the polynucleic acid molecule comprises the artificialnucleotide analogues described herein. In some instances, about 100% ofthe polynucleic acid molecule comprises the artificial nucleotideanalogues described herein. In some embodiments, the artificialnucleotide analogues include 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE),2′-O-aminopropyl, 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), or 2′-O—N-methylacetamido (2′-O-NMA) modified, LNA, ENA,PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonatenucleotides, 2′-fluoro N3-P5′-phosphoramidites, or a combinationthereof.

In some embodiments, the polynucleic acid molecule comprises from about1 to about 25 modifications in which the modification comprises anartificial nucleotide analogues described herein. In some embodiments,the polynucleic acid molecule comprises about 1 modification in whichthe modification comprises an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 2 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 3 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 4 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 5 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 6 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 7 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 8 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 9 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 10 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 11 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 12 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 13 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 14 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 15 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 16 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 17 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 18 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 19 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 20 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 21 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 22 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 23 modifications in which themodifications comprise an artificial nucleotide analogue describedherein. In some embodiments, the polynucleic acid molecule comprisesabout 24 modifications in which the modifications comprise an artificialnucleotide analogue described herein. In some embodiments, thepolynucleic acid molecule comprises about 25 modifications in which themodifications comprise an artificial nucleotide analogue describedherein.

In some embodiments, a polynucleic acid molecule is assembled from twoseparate polynucleotides wherein one polynucleotide comprises the sensestrand and the second polynucleotide comprises the antisense strand ofthe polynucleic acid molecule. In other embodiments, the sense strand isconnected to the antisense strand via a linker molecule, which in someinstances is a polynucleotide linker or a non-nucleotide linker.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein pyrimidine nucleotides in the sensestrand comprises 2′-O-methylpyrimidine nucleotides and purinenucleotides in the sense strand comprise 2′-deoxy purine nucleotides. Insome embodiments, a polynucleic acid molecule comprises a sense strandand antisense strand, wherein pyrimidine nucleotides present in thesense strand comprise 2′-deoxy-2′-fluoro pyrimidine nucleotides andwherein purine nucleotides present in the sense strand comprise 2′-deoxypurine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein the pyrimidine nucleotides whenpresent in said antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides when present in said antisensestrand are 2′-O-methyl purine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein the pyrimidine nucleotides whenpresent in said antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and wherein the purine nucleotides when present in saidantisense strand comprise 2′-deoxy-purine nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, and at least one of sense strand andantisense strands has a plurality of (e.g., two or more, three or more,four or more, five or more, six or more, seven or more, eight or more,etc) 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides. In someembodiments, where at least two out of the a plurality of 2′-O-methyl or2′-deoxy-2′-fluoro modified nucleotides are consecutive nucleotides. Insome embodiments, where consecutive 2′-O-methyl or 2′-deoxy-2′-fluoromodified nucleotides are located at the 5′-end of the sense strandand/or the antisense strand. In some embodiments, where consecutive2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides are located atthe 3′-end of the sense strand and/or the antisense strand. In someembodiments, the sense strand of polynucleic acid molecule includes atleast four, at least five, at least six consecutive 2′-O-methyl modifiednucleotides at its 5′ end and/or 3′end, or both. Optionally, in suchembodiments, the sense strand of polynucleic acid molecule includes atleast one, at least two, at least three, at least four2′-deoxy-2′-fluoro modified nucleotides at the 3′ end of the at leastfour, at least five, at least six consecutive 2′-O-methyl modifiednucleotides at the polynucleotides' 5′ end, or at the 5′ end of the atleast four, at least five, at least six consecutive 2′-O-methyl modifiednucleotides at polynucleotides' 3′ end. Also optionally, such at leasttwo, at least three, at least four 2′-deoxy-2′-fluoro modifiednucleotides are consecutive nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, and at least one of sense strand andantisense strands has 2′-O-methyl modified nucleotide located at the5′-end of the sense strand and/or the antisense strand. In someembodiments, at least one of sense strand and antisense strands has2′-O-methyl modified nucleotide located at the 3′-end of the sensestrand and/or the antisense strand. In some embodiments, the 2′-O-methylmodified nucleotide located at the 5′-end of the sense strand and/or theantisense strand is a purine nucleotide. In some embodiments, the2′-O-methyl modified nucleotide located at the 5′-end of the sensestrand and/or the antisense strand is a pyridine nucleotide.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, and the antisense strand has two or moreconsecutive 2′-deoxy-2′-fluoro modified nucleotides at 5′-end. In someembodiments, a polynucleic acid molecule comprises a sense strand andantisense strand, and the antisense strand has two or more consecutive2′-O-methyl modified nucleotides at 3′-end. In some embodiments, apolynucleic acid molecule comprises a sense strand and antisense strand,and the antisense strand has at least 2, 3, 4, 5, 6, or 7 consecutive2′-O-methyl modified nucleotides.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, and the sense strand comprises a nucleicacid of 5′-nsnsnnnnNfNfNfnnnnnnnnsnsa-3′ (lower case (n)=2′-O-Me(methyl), Nf=2′-F (fluoro); s=phosphorothioate backbone modification).In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, and the antisense strand comprises anucleic acid of 5′-UfsNfsnnnNfnnnnnnnNfnNfnnnsusu-3′ (lower case(n)=2′-O-Me (methyl), Nf=2′-F (fluoro); s=phosphorothioate backbonemodification). In some embodiments, a polynucleic acid moleculecomprises a sense strand and antisense strand, and the sense strandcomprises a nucleic acid of 5′-nsnsnnnnNfNfNfnnnnnnnnsnsa-3′ (lower case(n)=2′-O-Me (methyl), Nf=2′-F (fluoro); s=phosphorothioate backbonemodification) and the antisense strand comprises a nucleic acid of5′-UfsNfsnnnNfnnnnnnnNfnNfnnnsusu-3′ (lower case (n)=2′-O-Me (methyl),Nf=2′-F (fluoro); s=phosphorothioate backbone modification).

In some embodiments, a polynucleic acid molecule comprises a sensestrand and antisense strand, wherein the sense strand includes aterminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ends of the sense strand. In other embodiments, the terminal cap moietyis an inverted deoxy abasic moiety.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, wherein the antisense strand comprises aglyceryl modification at the 3′ end of the antisense strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the sense strand comprises oneor more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotidelinkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and in which the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. In otherembodiments, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more, pyrimidine nucleotides of the sense and/or antisense strandare chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the sense strand comprisesabout 1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and in which the antisense strand comprises about 1 toabout 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In other embodiments, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides ofthe sense and/or antisense strand are chemically-modified with 2′-deoxy,2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, with or without about1 to about 25 or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the antisense strand comprisesone or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotidelinkages, and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one ormore (e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the sense strand and/or antisense strand, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the sense strand. In some embodiments, the antisense strandcomprises about 1 to about 10 or more, specifically about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. In otherembodiments, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more pyrimidinenucleotides of the sense and/or antisense strand are chemically-modifiedwith 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoro nucleotides, withor without one or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more phosphorothioate internucleotide linkages and/or a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends, beingpresent in the same or different strand.

In some embodiments, a polynucleic acid molecule comprises a sensestrand and an antisense strand, in which the antisense strand comprisesabout 1 to about 25 or more, for example, about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and the antisense strand comprises about 1 to about 25or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or more phosphorothioate internucleotidelinkages, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the antisensestrand. In other embodiments, one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides of the sense and/orantisense strand are chemically-modified with 2′-deoxy, 2′-O-methyland/or 2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about5, for example about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In some embodiments, a polynucleic acid molecule described herein is achemically-modified short interfering nucleic acid molecule having about1 to about 25, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate internucleotidelinkages in each strand of the polynucleic acid molecule. In someembodiments, a polynucleic acid molecule comprises a sense strand and anantisense strand, and the antisense strand comprises a phosphatebackbone modification at the 3′ end of the antisense strand.Alternatively and/or additionally, a polynucleic acid molecule comprisesa sense strand and an antisense strand, and the sense strand comprises aphosphate backbone modification at the 5′ end of the antisense strand.In some instances, the phosphate backbone modification is aphosphorothioate. In some embodiments, the sense or antisense strand hasthree consecutive nucleosides that are coupled via two phosphorothioatebackbone.

In another embodiment, a polynucleic acid molecule described hereincomprises 2′-5′ internucleotide linkages. In some instances, the 2′-5′internucleotide linkage(s) is at the 3′-end, the 5′-end, or both of the3′- and 5′-ends of one or both sequence strands. In addition instances,the 2′-5′ internucleotide linkage(s) is present at various otherpositions within one or both sequence strands, for example, about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkageof a pyrimidine nucleotide in one or both strands of the polynucleicacid molecule comprise a 2′-5′ internucleotide linkage, or about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more including every internucleotide linkageof a purine nucleotide in one or both strands of the polynucleic acidmolecule comprise a 2′-5′ internucleotide linkage.

In some embodiments, a polynucleic acid molecule is a single strandedpolynucleic acid molecule that mediates RNAi activity in a cell orreconstituted in vitro system, wherein the polynucleic acid moleculecomprises a single stranded polynucleotide having complementarity to atarget nucleic acid sequence, and wherein one or more pyrimidinenucleotides present in the polynucleic acid are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and wherein any purine nucleotides present in the polynucleic acid are2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are2′-deoxy purine nucleotides or alternately a plurality of purinenucleotides are 2′-deoxy purine nucleotides), and a terminal capmodification, that is optionally present at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the antisense sequence, the polynucleicacid molecule optionally further comprising about 1 to about 4 (e.g.,about 1, 2, 3, or 4) terminal 2′-deoxynucleotides at the 3′-end of thepolynucleic acid molecule, wherein the terminal nucleotides furthercomprise one or more (e.g., 1, 2, 3, or 4) phosphorothioateinternucleotide linkages, and wherein the polynucleic acid moleculeoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group.

In some cases, one or more of the artificial nucleotide analoguesdescribed herein are resistant toward nucleases such as for exampleribonuclease such as RNase H, deoxyribonuclease such as DNase, orexonuclease such as 5′-3′ exonuclease and 3′-5′ exonuclease whencompared to natural polynucleic acid molecules. In some instances,artificial nucleotide analogues comprising 2′-O-methyl,2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, 2′-fluoroN3-P5′-phosphoramidites, or combinations thereof are resistant towardnucleases such as for example ribonuclease such as RNase H,deoxyribonuclease such as DNase, or exonuclease such as 5′-3′exonuclease and 3′-5′ exonuclease. In some instances, 2′-O-methylmodified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, 2′O-methoxyethyl (2′-O-MOE) modified polynucleic acidmolecule is nuclease resistance (e.g., RNase H, DNase, 5′-3′ exonucleaseor 3′-5′ exonuclease resistance). In some instances, 2′-O-aminopropylmodified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, 2′-deoxy modified polynucleic acid molecule is nucleaseresistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, 2′-deoxy-2′-fluoro modified polynucleicacid molecule is nuclease resistance (e.g., RNase H. DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). In some instances,2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule isnuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). In some instances, 2′-O-dimethylaminoethyl(2′-O-DMAOE) modified polynucleic acid molecule is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, 2′-O-dimethylaminopropyl (2′-O-DMAP)modified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modifiedpolynucleic acid molecule is nuclease resistance (e.g. RNase H, DNase,5′-3′ exonuclease or 3′-5′ exonuclease resistance). In some instances,2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acid molecule isnuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). In some instances, LNA modified polynucleicacid molecule is nuclease resistance (e.g., RNase H, DNase, 5′-3′exonuclease or 3′-5′ exonuclease resistance). In some instances, ENAmodified polynucleic acid molecule is nuclease resistance (e.g., RNaseH, DNase, 5′-3′ exonuclease or 3′-5′ exonuclease resistance). In someinstances, HNA modified polynucleic acid molecule is nuclease resistance(e.g., RNase H. DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, morpholinos is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, PNA modified polynucleic acid moleculeis resistant to nucleases (e.g., RNase H, DNase, 5′-3′ exonuclease or3′-5′ exonuclease resistance). In some instances, methylphosphonatenucleotides modified polynucleic acid molecule is nuclease resistance(e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′ exonucleaseresistance). In some instances, thiolphosphonate nucleotides modifiedpolynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase,5′-3′ exonuclease or 3′-5′ exonuclease resistance). In some instances,polynucleic acid molecule comprising 2′-fluoro N3-P5′-phosphoramiditesis nuclease resistance (e.g., RNase H, DNase, 5′-3′ exonuclease or 3′-5′exonuclease resistance). In some instances, the 5′ conjugates describedherein inhibit 5′-3′ exonucleolytic cleavage. In some instances, the 3′conjugates described herein inhibit 3′-5′ exonucleolytic cleavage.

In some embodiments, one or more of the artificial nucleotide analoguesdescribed herein have increased binding affinity toward their mRNAtarget relative to an equivalent natural polynucleic acid molecule. Theone or more of the artificial nucleotide analogues comprising2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy,2′-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP),2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or 2′-O—N-methylacetamido(2′-O-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonatenucleotides, thiolphosphonate nucleotides, or 2′-fluoroN3-P5′-phosphoramidites have increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid molecule.In some instances, 2′-O-methyl modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-methoxyethyl (2′-O-MOE) modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-aminopropyl modified polynucleic acid molecule has increasedbinding affinity toward their mRNA target relative to an equivalentnatural polynucleic acid molecule. In some instances, 2′-deoxy modifiedpolynucleic acid molecule has increased binding affinity toward theirmRNA target relative to an equivalent natural polynucleic acid molecule.In some instances, 2′-deoxy-2′-fluoro modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-aminopropyl (2′-O-AP) modified polynucleic acid molecule hasincreased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-dimethylaminoethyl (2′-O-DMAOE) modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-dimethylaminopropyl (2′-O-DMAP) modified polynucleic acid moleculehas increased binding affinity toward their mRNA target relative to anequivalent natural polynucleic acid molecule. In some instances,2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE) modified polynucleic acidmolecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, 2′-O—N-methylacetamido (2′-O-NMA) modified polynucleic acidmolecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, LNA modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances. ENA modified polynucleicacid molecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, PNA modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, HNA modified polynucleicacid molecule has increased binding affinity toward their mRNA targetrelative to an equivalent natural polynucleic acid molecule. In someinstances, morpholino modified polynucleic acid molecule has increasedbinding affinity toward their mRNA target relative to an equivalentnatural polynucleic acid molecule. In some instances, methylphosphonatenucleotides modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, thiolphosphonatenucleotides modified polynucleic acid molecule has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some instances, polynucleic acid moleculecomprising 2′-fluoro N3-P5′-phosphoramidites has increased bindingaffinity toward their mRNA target relative to an equivalent naturalpolynucleic acid molecule. In some cases, the increased affinity isillustrated with a lower Kd, a higher melt temperature (Tm), or acombination thereof.

In some embodiments, a polynucleic acid molecule described herein is achirally pure (or stereo pure) polynucleic acid molecule, or apolynucleic acid molecule comprising a single enantiomer. In someinstances, the polynucleic acid molecule comprises L-nucleotide. In someinstances, the polynucleic acid molecule comprises D-nucleotides. Insome instance, a polynucleic acid molecule composition comprises lessthan 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirrorenantiomer. In some cases, a polynucleic acid molecule compositioncomprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or lessof a racemic mixture. In some instances, the polynucleic acid moleculeis a polynucleic acid molecule described in: U.S. Patent PublicationNos: 2014/194610 and 2015/211006; and PCT Publication No: WO2015107425.

In some embodiments, a polynucleic acid molecule described herein isfurther modified to include an aptamer conjugating moiety. In someinstances, the aptamer conjugating moiety is a DNA aptamer conjugatingmoiety. In some instances, the aptamer conjugating moiety is Alphamer(Centauri Therapeutics), which comprises an aptamer portion thatrecognizes a specific cell-surface target and a portion that presents aspecific epitopes for attaching to circulating antibodies. In someinstance, a polynucleic acid molecule described herein is furthermodified to include an aptamer conjugating moiety as described in: U.S.Pat. Nos. 8,604,184, 8,591,910, and 7,850,975.

In additional embodiments, a polynucleic acid molecule described hereinis modified to increase its stability. In some embodiment, thepolynucleic acid molecule is RNA (e.g., siRNA). In some instances, thepolynucleic acid molecule is modified by one or more of themodifications described above to increase its stability. In some cases,the polynucleic acid molecule is modified at the 2′ hydroxyl position,such as by 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl,2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP),2′-0-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA) modification or by a locked or bridgedribose conformation (e.g., LNA or ENA). In some cases, the polynucleicacid molecule is modified by 2′-O-methyl and/or 2′-O-methoxyethylribose. In some cases, the polynucleic acid molecule also includesmorpholinos, PNAs, HNA, methylphosphonate nucleotides, thiolphosphonatenucleotides, and/or 2′-fluoro N3-P5′-phosphoramidites to increase itsstability. In some instances, the polynucleic acid molecule is achirally pure (or stereo pure) polynucleic acid molecule. In someinstances, the chirally pure (or stereo pure) polynucleic acid moleculeis modified to increase its stability. Suitable modifications to the RNAto increase stability for delivery will be apparent to the skilledperson.

In some instances, the polynucleic acid molecule is a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. In some instances, the polynucleic acid molecule isassembled from two separate polynucleotides, where one strand is thesense strand and the other is the antisense strand, wherein theantisense and sense strands are self-complementary (e.g., each strandcomprises nucleotide sequence that is complementary to nucleotidesequence in the other strand; such as where the antisense strand andsense strand form a duplex or double stranded structure, for examplewherein the double stranded region is about 19, 20, 21, 22, 23, or morebase pairs); the antisense strand comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense strand comprises nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.Alternatively, the polynucleic acid molecule is assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions of the polynucleic acid molecule are linked by means of anucleic acid based or non-nucleic acid-based linker(s).

In some cases, the polynucleic acid molecule is a polynucleotide with aduplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. In other cases, the polynucleic acid molecule is a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide is processed either in vivo or in vitro togenerate an active polynucleic acid molecule capable of mediating RNAi.In additional cases, the polynucleic acid molecule also comprises asingle-stranded polynucleotide having nucleotide sequence complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof (for example, where such polynucleic acid molecule does notrequire the presence within the polynucleic acid molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single stranded polynucleotide further comprises aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′, 3′-diphosphate.

In some instances, an asymmetric hairpin is a linear polynucleic acidmolecule comprising an antisense region, a loop portion that comprisesnucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complimentary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin polynucleic acid molecule comprises an antisense region havinglength sufficient to mediate RNAi in a cell or in vitro system (e.g.about 19 to about 22 nucleotides) and a loop region comprising about 4to about 8 nucleotides, and a sense region having about 3 to about 18nucleotides that are complementary to the antisense region. In somecases, the asymmetric hairpin polynucleic acid molecule also comprises a5′-terminal phosphate group that is chemically modified. In additionalcases, the loop portion of the asymmetric hairpin polynucleic acidmolecule comprises nucleotides, non-nucleotides, linker molecules, orconjugate molecules.

In some embodiments, an asymmetric duplex is a polynucleic acid moleculehaving two separate strands comprising a sense region and an antisenseregion, wherein the sense region comprises fewer nucleotides than theantisense region to the extent that the sense region has enoughcomplimentary nucleotides to base pair with the antisense region andform a duplex. For example, an asymmetric duplex polynucleic acidmolecule comprises an antisense region having length sufficient tomediate RNAi in a cell or in vitro system (e.g., about 19 to about 22nucleotides) and a sense region having about 3 to about 18 nucleotidesthat are complementary to the antisense region.

In some cases, a universal base refers to nucleotide base analogs thatform base pairs with each of the natural DNA/RNA bases with littlediscrimination between them. Non-limiting examples of universal basesinclude C-phenyl, C-naphthyl and other aromatic derivatives, inosine,azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole,4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (seefor example Loakes, 2001, Nucleic Acids Research. 29, 2437-2447).

Polynucleic Acid Molecule Synthesis

In some embodiments, a polynucleic acid molecule described herein isconstructed using chemical synthesis and/or enzymatic ligation reactionsusing procedures known in the art. For example, a polynucleic acidmolecule is chemically synthesized using naturally occurring nucleotidesor variously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the polynucleic acid molecule and target nucleicacids. Exemplary methods include those described in: U.S. Pat. Nos.5,142,047; 5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCTPublication No. WO2009099942; or European Publication NO. 1579015.Additional exemplary methods include those described in: Griffey et al.,“2′-O-aminopropyl ribonucleotides: a zwitterionic modification thatenhances the exonuclease resistance and biological activity of antisenseoligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al.“Synthesis of 2′-0,4′-C-methyleneuridine and -cytidine. Novel bicyclicnucleosides having a fixed C3, -endo sugar puckering”. TetrahedronLetters 38 (50): 8735 (1997); Koizumi, M. “ENA oligonucleotides astherapeutics”. Current opinion in molecular therapeutics 8 (2): 144-149(2006); and Abramova et al., “Novel oligonucleotide analogues based onmorpholino nucleoside subunits-antisense technologies: new chemicalpossibilities,” Indian Journal of Chemistry 48B:1721-1726 (2009).Alternatively, the polynucleic acid molecule is produced biologicallyusing an expression vector into which a polynucleic acid molecule hasbeen subcloned in an antisense orientation (i.e., RNA transcribed fromthe inserted polynucleic acid molecule will be of an antisenseorientation to a target polynucleic acid molecule of interest).

In some embodiments, a polynucleic acid molecule is synthesized via atandem synthesis methodology, wherein both strands are synthesized as asingle contiguous oligonucleotide fragment or strand separated by acleavable linker which is subsequently cleaved to provide separatefragments or strands that hybridize and permit purification of theduplex.

In some instances, a polynucleic acid molecule is also assembled fromtwo distinct nucleic acid strands or fragments wherein one fragmentincludes the sense region and the second fragment includes the antisenseregion of the molecule.

Additional modification methods for incorporating, for example, sugar,base and phosphate modifications include: Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature. 1990, 344,565-568; Pieken et al. Science. 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. IntemationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Let., 39, 1131; Eamshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010. Such publications describe general methods and strategies todetermine the location of incorporation of sugar, base and/or phosphatemodifications and the like into nucleic acid molecules withoutmodulating catalysis.

In some instances, while chemical modification of the polynucleic acidmolecule internucleotide linkages with phosphorothioate,phosphorodithioate, and/or 5′-methylphosphonate linkages improvesstability, excessive modifications sometimes cause toxicity or decreasedactivity. Therefore, when designing nucleic acid molecules, the amountof these internucleotide linkages in some cases is minimized. In suchcases, the reduction in the concentration of these linkages lowerstoxicity, increases efficacy and higher specificity of these molecules.

Polynucleic Acid Molecule Conjugates

In some embodiments, a polynucleic acid molecule (B) is furtherconjugated to a polypeptide (A) for delivery to a site of interest. Insome instances, at least one polypeptide A is conjugated to at least oneB. In some instances, the at least one polypeptide A is conjugated tothe at least one B to form an A-B conjugate. In some embodiments, atleast one A is conjugated to the 5′ terminus of B, the 3′ terminus of B,an internal site on B, or in any combinations thereof. In someinstances, the at least one polypeptide A is conjugated to at least twoB. In some instances, the at least one polypeptide A is conjugated to atleast 2, 3, 4, 5, 6, 7, 8, or more B.

In some cases, a polynucleic acid molecule is conjugated to apolypeptide (A) and optionally a polymeric moiety (C). In someembodiments, at least one polypeptide A is conjugated at one terminus ofat least one B while at least one C is conjugated at the oppositeterminus of the at least one B to form an A-B-C conjugate. In someinstances, at least one polypeptide A is conjugated at one terminus ofthe at least one B while at least one of C is conjugated at an internalsite on the at least one B. In some instances, at least one polypeptideA is conjugated directly to the at least one C. In some instances, theat least one B is conjugated indirectly to the at least one polypeptideA via the at least one C to form an A-C-B conjugate.

In some instances, at least one B and/or at least one C. and optionallyat least one D are conjugated to at least one polypeptide A. In someinstances, the at least one B is conjugated at a terminus (e.g., a 5′terminus or a 3′ terminus) to the at least one polypeptide A or areconjugated via an internal site to the at least one polypeptide A. Insome cases, the at least one C is conjugated either directly to the atleast one polypeptide A or indirectly via the at least one B. Ifindirectly via the at least one B, the at least one C is conjugatedeither at the same terminus as the at least one polypeptide A on B, atopposing terminus from the at least one polypeptide A, or independentlyat an internal site. In some instances, at least one additionalpolypeptide A is further conjugated to the at least one polypeptide A,to B, or to C. In additional instances, the at least one D is optionallyconjugated either directly or indirectly to the at least one polypeptideA, to the at least one B, or to the at least one C. If directly to theat least one polypeptide A, the at least one D is also optionallyconjugated to the at least one B to form an A-D-B conjugate or isoptionally conjugated to the at least one B and the at least one C toform an A-D-B-C conjugate. In some instances, the at least one D isdirectly conjugated to the at least one polypeptide A and indirectly tothe at least one B and the at least one C to form a D-A-B-C conjugate.If indirectly to the at least one polypeptide A, the at least one D isalso optionally conjugated to the at least one B to form an A-B-Dconjugate or is optionally conjugated to the at least one B and the atleast one C to form an A-B-D-C conjugate. In some instances, at leastone additional D is further conjugated to the at least one polypeptideA, to B, or to C.

Binding Moiety

In some embodiments, the binding moiety A is a polypeptide. In someinstances, the polypeptide is an antibody or its fragment thereof. Insome cases, the fragment is a binding fragment. In some instances, theantibody or antigen binding fragment thereof comprises a humanizedantibody or antigen binding fragment thereof, murine antibody or antigenbinding fragment thereof, chimeric antibody or antigen binding fragmentthereof, monoclonal antibody or antigen binding fragment thereof, abinding fragment having a light chain domain and a heavy chain domain, abinding fragment having two light chain domains and two heavy chaindomains, a binding fragment having two or more light chain domains andheavy chain domains, monovalent Fab′, divalent Fab₂, F(ab)′₃ fragments,single-chain variable fragment (scFv), bis-scFv, (scFv)₂, diabody,minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein(dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody orantigen binding fragment thereof, bispecific antibody or biding fragmentthereof, or a chemically modified derivative thereof.

In some embodiments, the binding moiety A is a bispecific antibody orantigen binding fragment thereof. In some instances, the bispecificantibody is a trifunctional antibody or a bispecific mini-antibody. Insome cases, the bispecific antibody is a trifunctional antibody. In someinstances, the trifunctional antibody is a full length monoclonalantibody comprising binding sites for two different antigens.

In some cases, the bispecific antibody is a bispecific mini-antibody. Insome instances, the bispecific mini-antibody comprises divalent Fab₂,F(ab)'₃ fragments, bis-scFv, (scFv)₂, diabody, minibody, triabody,tetrabody or a bi-specific T-cell engager (BiTE). In some embodiments,the bi-specific T-cell engager is a fusion protein that contains twosingle-chain variable fragments (scFvs) in which the two scFvs targetepitopes of two different antigens.

In some embodiments, the binding moiety A is a bispecific mini-antibody.In some instances, A is a bispecific Fab₂. In some instances, A is abispecific F(ab)′₃ fragment. In some cases, A is a bispecific bis-scFv.In some cases, A is a bispecific (scFv)₂. In some embodiments, A is abispecific diabody. In some embodiments, A is a bispecific minibody. Insome embodiments. A is a bispecific triabody. In other embodiments, A isa bispecific tetrabody. In other embodiments, A is a bi-specific T-cellengager (BiTE).

In some embodiments, the binding moiety A is a trispecific antibody. Insome instances, the trispecific antibody comprises F(ab)′₃ fragments ora triabody. In some instances, A is a trispecific F(ab)′₃ fragment. Insome cases, A is a triabody. In some embodiments, A is a trispecificantibody as described in Dimas, et al., “Development of a trispecificantibody designed to simultaneously and efficiently target threedifferent antigens on tumor cells,” Mol. Pharmaceutics, 12(9): 3490-3501(2015).

In some embodiments, the binding moiety A is an antibody or antigenbinding fragment thereof that recognizes a cell surface protein. In someinstances, the binding moiety A is an antibody or antigen bindingfragment thereof that recognizes a cell surface protein on a musclecell. In some cases, the binding moiety A is an antibody or antigenbinding fragment thereof that recognizes a cell surface protein on askeletal muscle cell.

In some embodiments, exemplary antibodies include, but are not limitedto, an anti-myosin antibody, an anti-transferrin receptor antibody, andan antibody that recognizes Muscle-Specific kinase (MuSK). In someinstances, the antibody is an anti-transferrin receptor (anti-CD71)antibody.

In some embodiments, where the antibody is an anti-transferrin receptor(anti-CD71) antibody, the anti-transferrin antibody specifically bindsto a transferrin receptor (TfR), preferably, specifically binds totransferrin receptor 1 (TfR1), or more preferably, specifically binds tohuman transferrin receptor 1 (TfR1) (or human CD71).

In some instances, the anti-transferrin receptor antibody comprises avariable heavy chain (VH) region and a variable light chain (VL) region,wherein the VH region comprises HCDR1 sequence comprising SEQ ID NO:281; HCDR2 sequence EINPIX₁GRSNYAX₂KFQG (SEQ ID NO: 406), wherein X₁ isselected from N or Q and X₂ is selected from Q or E; and HCDR3 sequencecomprising SEQ ID NO: 283.

In some embodiments, the VH region of the anti-transferrin receptorantibody comprises HCDR1, HCDR2, and HCDR3 sequences selected from Table1.

TABLE 1 SEQ SEQ SEQ ID ID ID Name HCDR1 NO: HCDR2 NO: HCDR3 NO: 13E4_VH1YTFTNYWMH 281 EINPINGRSN 282 GTRAMHY 283 YAQKFQG 13E4_VH2* YTFTNYWMH 281EINPINGRSN 284 GTRAMHY 283 YAEKFQG 13E4_VH3 YTFTNYWMH 281 EINPIQGRSN 285GTRAMHY 283 YAEKFQG *13E4_VH2 shares the same HCDR1, HCDR2, and HCDR3sequences with anti-transferrin receptor antibody 13E4_VH4

In some embodiments, the VH region comprises HCDR1 sequence comprisingSEQ ID NO: 281; HCDR2 sequence comprising SEQ ID NO: 282, 284, or 285;and HCDR3 sequence comprising SEQ ID NO: 283. In some instances, the VHregion comprises HCDR1 sequence comprising SEQ ID NO: 281, HCDR2sequence comprising SEQ ID NO: 282, and HCDR3 sequence comprising SEQ IDNO: 283. In some instances, the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 284, andHCDR3 sequence comprising SEQ ID NO: 283. In some instances, the VHregion comprises HCDR1 sequence comprising SEQ ID NO: 281, HCDR2sequence comprising SEQ ID NO: 285, and HCDR3 sequence comprising SEQ IDNO: 283.

In some embodiments, the VL region of the anti-transferrin receptorantibody comprises LCDR1 sequence RTSENIYX₃NLA (SEQ ID NO: 407), LCDR2sequence AX₄TNLAX₅ (SEQ ID NO: 408), and LCDR3 sequence QHFWGTPLTX₆ (SEQID NO: 409), wherein X₃ is selected from N or S. X₄ is selected from Aor G, X₅ is selected from D or E, and X₆ is present or absence, and ifpresent, is F.

In some embodiments, the VL region of the anti-transferrin receptorantibody comprises LCDR1, LCDR2, and LCDR3 sequences selected from Table2.

TABLE 2 SEQ SEQ SEQ ID ID ID Name LCDR1 NO: LCDR2 NO: LCDR3 NO:13E4_VL1* RTSENIYNNLA 286 AATNLAD 287 QHFWGTPLT 288 13E4 VL3 RTSENIYNNLA286 AATNLAE 289 QHFWGTPLTF 290 13E4_VL4 RTSENIYSNLA 291 AGTNLAD 292QHFWGTPLTF 290 *13E4_VL1 shares the same LCDR1, LCDR2, and LCDR3sequences with anti-transferrin receptor antibody 13E4_VL2

In some instances, the VL region comprises LCDR1 sequence RTSENIYX₃NLA(SEQ ID NO: 407), LCDR2 sequence comprising SEQ ID NO: 287, 289, or 292,and LCDR3 sequence comprising SEQ ID NO: 288 or 290, wherein X₃ isselected from N or S.

In some instances, the VL region comprises LCDR1 sequence comprising SEQID NO: 286 or 291, LCDR2 sequence AX₄TNLAX₅ (SEQ ID NO: 408), and LCDR3sequence comprising SEQ ID NO: 288 or 290, wherein X₄ is selected from Aor G, and X₅ is selected from D or E.

In some instances, the VL region comprises LCDR1 sequence comprising SEQID NO: 286 or 291, LCDR2 sequence SEQ ID NO: 287, 289, or 292, and LCDR3sequence QHFWGTPLTX₆ (SEQ ID NO: 409), wherein X₆ is present or absence,and if present, is F.

In some instances, the VL region comprises LCDR1 sequence comprising SEQID NO: 286, LCDR2 sequence AATNLAX₅ (SEQ ID NO: 410), and LCDR3 sequenceQHFWGTPLTX₆ (SEQ ID NO: 409), wherein X₅ is selected from D or E and X₆is present or absence, and if present, is F.

In some instances, the VL region comprises LCDR1 sequence comprising SEQID NO: 286, LCDR2 sequence comprising SEQ ID NO: 287, and LCDR3 sequencecomprising SEQ ID NO: 288.

In some instances, the VL region comprises LCDR1 sequence comprising SEQID NO: 286, LCDR2 sequence comprising SEQ ID NO: 289, and LCDR3 sequencecomprising SEQ ID NO: 290.

In some instances, the VL region comprises LCDR1 sequence comprising SEQID NO: 291, LCDR2 sequence comprising SEQ ID NO: 292, and LCDR3 sequencecomprising SEQ ID NO: 290.

In some embodiments, the anti-transferrin receptor antibody comprises aVH region and a VL region, wherein the VH region comprises HCDR1sequence comprising SEQ ID NO: 281: HCDR2 sequence EINPIX₁GRSNYAX₂KFQG(SEQ ID NO: 406), wherein X₁ is selected from N or Q and X₂ is selectedfrom Q or E; and HCDR3 sequence comprising SEQ ID NO: 283; and the VLregion comprises LCDR1 sequence RTSENIYX₃NLA (SEQ ID NO: 407), LCDR2sequence AX₄TNLAX₅ (SEQ ID NO: 408), and LCDR3 sequence QHFWGTPLTX₆ (SEQID NO: 409), wherein X₃ is selected from N or S, X₄ is selected from Aor G, X₅ is selected from D or E, and X₆ is present or absence, and ifpresent, is F.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, wherein the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281: HCDR2 sequence EINPIX₁GRSNYAX₂KFQG (SEQ IDNO: 406), wherein X₁ is selected from N or Q and X₂ is selected from Qor E; and HCDR3 sequence comprising SEQ ID NO: 283; and the VL regioncomprises LCDR1 sequence RTSENIYX₃NLA (SEQ ID NO: 407), LCDR2 sequencecomprising SEQ ID NO: 287, 289, or 292, and LCDR3 sequence comprisingSEQ ID NO: 288 or 290, wherein X₃ is selected from N or S.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, wherein the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281; HCDR2 sequence EINPIX₁GRSNYAX₂KFQG (SEQ IDNO: 406), wherein X₁ is selected from N or Q and X₂ is selected from Qor E; and HCDR3 sequence comprising SEQ ID NO: 283; and the VL regioncomprises LCDR1 sequence comprising SEQ ID NO: 286 or 291, LCDR2sequence AX₄TNLAX₅ (SEQ ID NO: 408), and LCDR3 sequence comprising SEQID NO: 288 or 290, w % herein X₄ is selected from A or G, and X₅ isselected from D or E.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, wherein the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281; HCDR2 sequence EINPIX₁GRSNYAX₂KFQG (SEQ IDNO: 406), wherein X₁ is selected from N or Q and X₂ is selected from Qor E; and HCDR3 sequence comprising SEQ ID NO: 283; and the VL regioncomprises LCDR1 sequence comprising SEQ ID NO: 286 or 291, LCDR2sequence SEQ ID NO: 287, 289, or 292, and LCDR3 sequence QHFWGTPLTX₆(SEQ ID NO: 409), wherein X₆ is present or absence, and if present, isF.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, wherein the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281; HCDR2 sequence EINPIX₁GRSNYAX₂KFQG (SEQ IDNO: 406), wherein X₁ is selected from N or Q and X₂ is selected from Qor E; and HCDR3 sequence comprising SEQ ID NO: 283; and the VL regioncomprises LCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequenceAATNLAX₅ (SEQ ID NO: 410), and LCDR3 sequence QHFWGTPLTX₆ (SEQ ID NO:409), wherein X₅ is selected from D or E and X₆ is present or absence,and if present, is F.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, wherein the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281; HCDR2 sequence EINPIX₁GRSNYAX₂KFQG (SEQ IDNO: 406), wherein X₁ is selected from N or Q and X₂ is selected from Qor E; and HCDR3 sequence comprising SEQ ID NO: 283; and the VL regioncomprises LCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequencecomprising SEQ ID NO: 287, and LCDR3 sequence comprising SEQ ID NO: 288.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, wherein the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281: HCDR2 sequence EINPIX₁GRSNYAX₂KFQG (SEQ IDNO: 406), wherein X₁ is selected from N or Q and X₂ is selected from Qor E; and HCDR3 sequence comprising SEQ ID NO: 283; and the VL regioncomprises LCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequencecomprising SEQ ID NO: 289, and LCDR3 sequence comprising SEQ ID NO: 290.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, wherein the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281: HCDR2 sequence EINPIX₁GRSNYAX₂KFQG (SEQ IDNO: 406), wherein X₁ is selected from N or Q and X₂ is selected from Qor E; and HCDR3 sequence comprising SEQ ID NO: 283, and the VL regioncomprises LCDR1 sequence comprising SEQ ID NO: 291, LCDR2 sequencecomprising SEQ ID NO: 292, and LCDR3 sequence comprising SEQ ID NO: 290.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 282, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence RTSENIYX₃NLA (SEQ ID NO: 407), LCDR2 sequence comprisingSEQ ID NO: 287, 289, or 292, and LCDR3 sequence comprising SEQ ID NO:288 or 290, wherein X₃ is selected from N or S.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 282, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286 or 291, LCDR2 sequenceAX₄TNLAX₅ (SEQ ID NO: 408), and LCDR3 sequence comprising SEQ ID NO: 288or 290, wherein X₄ is selected from A or G. and X₅ is selected from D orE.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 2, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286 or 291, LCDR2 sequence SEQ IDNO: 287, 289, or 292, and LCDR3 sequence QHFWGTPLTX₆ (SEQ ID NO: 409),wherein X₆ is present or absence, and if present, is F.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 282, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequence AATNLAX₅ (SEQID NO: 410), and LCDR3 sequence QHFWGTPLTX₆ (SEQ ID NO: 409), wherein X₅is selected from D or E and X₆ is present or absence, and if present, isF.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 282, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequence comprising SEQID NO: 287, and LCDR3 sequence comprising SEQ ID NO: 288.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 282, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequence comprising SEQID NO: 289, and LCDR3 sequence comprising SEQ ID NO: 290.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 282, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 291, LCDR2 sequence comprising SEQID NO: 292, and LCDR3 sequence comprising SEQ ID NO: 290.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 284, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence RTSENIYX₃NLA (SEQ ID NO: 407), LCDR2 sequence comprisingSEQ ID NO: 287, 289, or 292, and LCDR3 sequence comprising SEQ ID NO:288 or 290, wherein X₃ is selected from N or S.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 284, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286 or 291, LCDR2 sequenceAX₄TNLAX₅ (SEQ ID NO: 408), and LCDR3 sequence comprising SEQ ID NO: 288or 290, wherein X₄ is selected from A or G, and X₅ is selected from D orE.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 284, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286 or 291, LCDR2 sequence SEQ IDNO: 287, 289, or 292, and LCDR3 sequence QHFWGTPLTX₆ (SEQ ID NO: 409),wherein X₆ is present or absence, and if present, is F.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 284, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequence AATNLAX₅ (SEQID NO: 410), and LCDR3 sequence QHFWGTPLTX₆ (SEQ ID NO: 409), wherein X₅is selected from D or E and X₆ is present or absence, and if present, isF.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 284, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequence comprising SEQID NO: 287, and LCDR3 sequence comprising SEQ ID NO: 288.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO; 284, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequence comprising SEQID NO: 289, and LCDR3 sequence comprising SEQ ID NO:290.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 284, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 291, LCDR2 sequence comprising SEQID NO: 292, and LCDR3 sequence comprising SEQ ID NO: 290.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 285, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence RTSENIYX₃NLA (SEQ ID NO: 407), LCDR2 sequence comprisingSEQ ID NO: 287, 289, or 29, and LCDR3 sequence comprising SEQ ID NO: 288or 290, wherein X₃ is selected from N or S.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 285, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286 or 291, LCDR2 sequenceAX₄TNLAX₅ (SEQ ID NO: 408), and LCDR3 sequence comprising SEQ ID NO: 288or 290, wherein X₄ is selected from A or G, and X₅ is selected from D orE.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 285, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286 or 291, LCDR2 sequence SEQ IDNO: 287, 289, or 292, and LCDR3 sequence QHFWGTPLTX₆ (SEQ ID NO: 409),wherein X₆ is present or absence, and if present, is F.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 285, andHCDR3 sequence comprising SEQ ID NO: 283 and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequence AATNLAX₅ (SEQID NO: 410), and LCDR3 sequence QHFWGTPLTX₆ (SEQ ID NO: 409), wherein X₅is selected from D or E and X₆ is present or absence, and if present, isF.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 285, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequence comprising SEQID NO: 287, and LCDR3 sequence comprising SEQ ID NO: 288.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 285, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 286, LCDR2 sequence comprising SEQID NO: 289, and LCDR3 sequence comprising SEQ ID NO: 290.

In some instances, the anti-transferrin receptor antibody comprises a VHregion and a VL region, in which the VH region comprises HCDR1 sequencecomprising SEQ ID NO: 281, HCDR2 sequence comprising SEQ ID NO: 285, andHCDR3 sequence comprising SEQ ID NO: 283; and the VL region comprisesLCDR1 sequence comprising SEQ ID NO: 291, LCDR2 sequence comprising SEQID NO: 292, and LCDR3 sequence comprising SEQ ID NO: 290.

In some embodiments, the anti-transferrin receptor antibody comprises aVH region and a VL region in which the sequence of the VH regioncomprises about 80%, 85%, 90%, 95%, 96% 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 293-296 and the sequence of the VL regioncomprises about 80%, 85%, 90%, 95%, 96% 97%, 98%, 99%, or 100% sequenceidentity to SEQ ID NOs: 298-301.

In some embodiments, the VH region comprises a sequence selected fromSEQ ID NOs: 293-296 (Table 3) and the VL region comprises a sequenceselected from SEQ ID NOs: 298-301 (Table 4). The underlined regions inTable 3 and Table 4 denote the respective CDR1, CDR2, or CDR3 sequence.

TABLE 3 NAME VH SEQUENCE SEQ ID NO: 13E4_VH1QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVR 293QAPGQGLEWMGEINPINGRSNYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSS 13E4_VH2QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVR 294QAPGQGLEWIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSS 13E4_VH3QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVR 295QAPGQGLEWMGEINPIQGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSS 13E4_VH4QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVR 296QAPGQGLEWMGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSS 13E4_VHQVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMHWVKQ 297RPGQGLEWIGEINPINGRSNYGERFKTKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAMHYWGQGTSVTVSS

TABLE 4 NAME VL SEQUENCE SEQ ID NO: 13E4_VL1DIQMTQSPSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGK 298SPKLLIYAATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFAT YYCQHFWGTPLTFGGGTKVEIK13E4_VL2 DIQMTQSPSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGK 299APKLLIYAATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFAT YYCQHFWGTPLTFGGGTKVEIK13E4_VL3 DIQMTQSPSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGK 300APKLLIYAATNLAEGVPSRFSGSGSGTDYTLTISSLQPEDFAT YYCQHFWGTPLTFGGGTKVEIK13E4_VL4 DIQMTQSPSSLSASVGDRVTITCRTSENIYSNLAWYQQKPGK 301APKLLIYAGTNLADGVPSRFSGSGSGTDYTLTISSLQPEDFAN YYCQHFWGTPLTFGGGTKVEIK13E4_VL DIQMTQSPASLSVSVGETVTITCRTSENIYNNLAWYQQKQGK 302SPQLLVYAATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFG NYYCQHFWGTPLTFGAGTKLELK

In some embodiments, the anti-transferrin receptor antibody comprises aVH region and a VL region as illustrated in Table 5.

TABLE 5 13E4_VH1 13E4_VH2 13E4_VH3 13E4_VH4 (SEQ ID NO: 293) (SEQ ID NO:294) (SEQ ID NO: 295) (SEQ ID NO: 296) 13E4_VL1 SEQ ID NO: 293 + SEQ IDNO: 294 + SEQ ID NO: 295 + SEQ ID NO: 296 + (SEQ ID NO: 298) SEQ ID NO:298 SEQ ID NO: 298 SEQ ID NO: 298 SEQ ID NO: 298 13E4_VL2 SEQ ID NO:293 + SEQ ID NO: 294 + SEQ ID NO: 295 + SEQ ID NO: 296 + (SEQ ID NO:299) SEQ ID NO: 299 SEQ ID NO: 299 SEQ ID NO: 299 SEQ ID NO: 29913E4_VL3 SEQ ID NO: 293 + SEQ ID NO: 294 + SEQ ID NO: 295 + SEQ ID NO:296 + (SEQ ID NO: 300) SEQ ID NO: 300 SEQ ID NO: 300 SEQ ID NO: 300 SEQID NO: 300 13E4_VL4 SEQ ID NO: 293 + SEQ ID NO: 294 + SEQ ID NO: 295 +SEQ ID NO: 296 + (SEQ ID NO: 301) SEQ ID NO: 301 SEQ ID NO: 301 SEQ IDNO: 301 SEQ ID NO: 301

In some embodiments, an anti-transferrin receptor antibody describedherein comprises an IgG framework, an IgA framework, an IgE framework,or an IgM framework. In some instances, the anti-transferrin receptorantibody comprises an IgG framework (e.g., IgG1, IgG2, IgG3, or IgG4).In some cases, the anti-transferrin receptor antibody comprises an IgG1framework. In some cases, the anti-transferrin receptor antibodycomprises an IgG2 (e.g., an IgG2a or IgG2b) framework. In some cases,the anti-transferrin receptor antibody comprises an IgG2a framework. Insome cases, the anti-transferrin receptor antibody comprises an IgG2bframework. In some cases, the anti-transferrin receptor antibodycomprises an IgG3 framework. In some cases, the anti-transferrinreceptor antibody comprises an IgG4 framework.

In some cases, an anti-transferrin receptor antibody comprises one ormore mutations in a framework region, e.g., in the CH1 domain, CH2domain, CH3 domain, hinge region, or a combination thereof. In someinstances, the one or more mutations are to stabilize the antibodyand/or to increase half-life. In some instances, the one or moremutations are to modulate Fc receptor interactions, to reduce oreliminate Fc effector functions such as FcyR, antibody-dependentcell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity(CDC). In additional instances, the one or more mutations are tomodulate glycosylation.

In some embodiments, the one or more mutations are located in the Fcregion. In some instances, the Fc region comprises a mutation at residueposition L234, L235, or a combination thereof. In some instances, themutations comprise L234 and L235. In some instances, the mutationscomprise L234A and L235A. In some cases, the residue positions are inreference to IgG1.

In some instances, the Fc region comprises a mutation at residueposition L234, L235, D265, N297, K322, L328, or P329, or a combinationthereof. In some instances, the mutations comprise L234 and L235 incombination with a mutation at residue position K322, L328, or P329. Insome cases, the Fc region comprises mutations at L234. L235, and K322.In some cases, the Fc region comprises mutations at L234, L235, andL328. In some cases, the Fc region comprises mutations at L234. L235,and P329. In some cases, the Fc region comprises mutations at D265 andN297. In some cases, the residue position is in reference to IgGI.

In some instances, the Fc region comprises L234A. L235A, D265A, N297G,K322G, L328R, or P329G, or a combination thereof. In some instances, theFc region comprises L234A and L235A in combination with K322G, L328R, orP329G. In some cases, the Fc region comprises L234A, L235A, and K322G.In some cases, the Fc region comprises L234A, L235A, and L328R. In somecases, the Fc region comprises L234A, L235A, and P329G. In some cases,the Fc region comprises D265A and N297G. In some cases, the residueposition is in reference to IgG1.

In some instances, the Fc region comprises a mutation at residueposition L235, L236, D265, N297, K322, L328, or P329, or a combinationof the mutations. In some instances, the Fc region comprises mutationsat L235 and L236. In some instances, the Fc region comprises mutationsat L235 and L236 in combination with a mutation at residue positionK322, L328, or P329. In some cases, the Fc region comprises mutations atL235. L236, and K322. In some cases, the Fc region comprises mutationsat L235, L236, and L328. In some cases, the Fc region comprisesmutations at L235. L236, and P329. In some cases, the Fc regioncomprises mutations at D265 and N297. In some cases, the residueposition is in reference to IgG2b.

In some embodiments, the Fc region comprises L235A, L236A, D265A, N297G,K322G, L328R. or P329G, or a combination thereof. In some instances, theFc region comprises L235A and L236A. In some instances, the Fc regioncomprises L235A and L236A in combination with K322G, L328R, or P329G. Insome cases, the Fc region comprises L235A, L236A, and K322G. In somecases, the Fc region comprises L235A, L236A, and L328R. In some cases,the Fc region comprises L235A, L236A, and P329G. In some cases, the Fcregion comprises D265A and N297G. In some cases, the residue position isin reference to IgG2b.

In some embodiments, the Fc region comprises a mutation at residueposition L233, L234, D264, N296, K321, L327, or P328, wherein theresidues correspond to positions 233, 234, 264, 296, 321, 327, and 328of SEQ ID NO: 303. In some instances, the Fc region comprises mutationsat L233 and L234. In some instances, the Fc region comprises mutationsat L233 and L234 in combination with a mutation at residue positionK321, L327, or P328. In some cases, the Fc region comprises mutations atL233, L234, and K321. In some cases, the Fc region comprises mutationsat L233, L234, and L327. In some cases, the Fc region comprisesmutations at L233, L234, and K321. In some cases, the Fc regioncomprises mutations at L233, L234, and P328. In some instances, the Fcregion comprises mutations at D264 and N296. In some cases, equivalentpositions to residue L233, L234, D264, N296, K321, L327, or P328 in anIgG1, IgG2, IgG3, or IgG4 framework are contemplated. In some cases,mutations to a residue that corresponds to residue L233, L234, D264,N296, K321, L327, or P328 of SEQ ID NO: 23 in an IgG1, IgG2, or IgG4framework are also contemplated.

In some embodiments, the Fc region comprises L233A, L234A, D264A, N296G,K321G, L327R. or P328G, wherein the residues correspond to positions233, 234, 264, 296, 321, 327, and 328 of SEQ ID NO: 303. In someinstances, the Fc region comprises L233A and L234A. In some instances,the Fc region comprises L233A and L234A in combination with K321G,L327R, or P328G. In some cases, the Fc region comprises L233A, L234A,and K321G. In some cases, the Fc region comprises L233A. L234A, andL327R. In some cases, the Fc region comprises L233A, L234A, and K321G.In some cases, the Fc region comprises L233A, L234A, and P328G. In someinstances, the Fc region comprises D264A and N296G.

In some embodiments, the human IgG constant region is modified to alterantibody-dependent cellular cytotoxicity (ADCC) and/orcomplement-dependent cytotoxicity (CDC), e.g., with an amino acidmodification described in Natsume et al., 2008 Cancer Res, 68(10):3863-72; Idusogie et al., 2001 J Immunol, 166(4): 2571-5; Moore et al.,2010 mAbs, 2(2): 181-189: Lazar et al., 2006 PNAS, 103(11): 4005-4010,Shields et al., 2001 JBC, 276(9): 6591-6604; Stavenhagen et al., 2007Cancer Res. 67(18): 8882-8890; Stavenhagen et al., 2008 Advan. EnzymeRegul., 48: 152-164; Alegre et al, 1992 J Immunol, 148: 3461-3468;Reviewed in Kaneko and Niwa, 2011 Biodrugs, 25(1): 1-11.

In some embodiments, an anti-transferrin receptor antibody describedherein is a full-length antibody, comprising a heavy chain (HC) and alight chain (LC). In some cases, the heavy chain (HC) comprises asequence selected from Table 6. In some cases, the light chain (LC)comprises a sequence selected from Table 7. The underlined regiondenotes the respective CDRs.

TABLE 6 NAME HC SEQUENCE SEQ ID NO: 13E4_VH1QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 303APGQGLEWMGEINPINGRSNYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH1_aQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 304APGQGLEWMGEINPINGRSNYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH1_bQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 305APGQGLEWMGEINPINGRSNYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCGVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH1_cQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 306APGQGLEWMGEINPINGRSNYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E_VH1_dQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 307APGQGLEWMGEINPINGRSNYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH1_eQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 308APGQGLEWMGEINPINGRSNYAQKFQGRVTLTVDTSISTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH2QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 309APGQGLEWIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH2_aQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 310APGQGLEWIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH2_bQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 311APGQGLEWIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCGVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH2_cQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 312APGQGLEWIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH2_dQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 313APGQGLEWIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH2_eQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 314APGQGLEWIGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSRLRSDDTAVYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH3QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 315APGQGLEWMGEINPIQGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH3_aQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 316APGQGLEWMGEINPIQGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH3_bQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 317APGQGLEWMGEINPIQGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCGVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH3_cQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 318APGQGLEWMGEINPIQGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH3_dQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 319APGQGLEWMGEINPIQGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH3_eQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 320APGQGLEWMGEINPIQGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH4QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 321APGQGLEWMGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH4_aQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 322APGQGLEWMGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH4_bQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 323APGQGLEWMGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCGVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH4_cQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 324APGQGLEWMGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKARPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH4_dQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 325APGQGLEWMGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 13E4_VH4_eQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMHWVRQ 326APGQGLEWMGEINPINGRSNYAEKFQGRVTLTVDTSSSTAYMELSSLRSEDTATYYCARGTRAMHYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

TABLE 7 NAME LC SEQUENCE SEQ ID NO: 13E4_VL1DIQMTQSPSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGK 327SPKLLIYAATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 13E4_VL2DIQMTQSPSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGK 328APKLLIYAATNLADGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 13E4_VL3DIQMTQSPSSLSASVGDRVTITCRTSENIYNNLAWYQQKPGK 329APKLLIYAATNLAEGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQHFWGTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC 13E4_VL4DIQMTQSPSSLSASVGDRVTITCRTSENIYSNLAWYQQKPGK 330APKLLIYAGTNLADGVPSRFSGSGSGTDYTLTISSLQPEDFANYYCQHFWGTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC

In some embodiments, an anti-transferrin receptor antibody describedherein has an improved serum half-life compared to a referenceanti-transferrin receptor antibody. In some instances, the improvedserum half-life is at least 30 minutes, 1 hour, 1.5 hour, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 14 days, 30 days, or longer than reference anti-transferrinreceptor antibody.

In some embodiments, the binding moiety A is conjugated to a polynucleicacid molecule (B) non-specifically. In some instances, the bindingmoiety A is conjugated to a polynucleic acid molecule (B) via a lysineresidue or a cysteine residue, in a non-site specific manner. In someinstances, the binding moiety A is conjugated to a polynucleic acidmolecule (B) via a lysine residue (e.g., lysine residue present in thebinding moiety A) in a non-site specific manner. In some cases, thebinding moiety A is conjugated to a polynucleic acid molecule (B) via acysteine residue (e.g., cysteine residue present in the binding moietyA) in a non-site specific manner.

In some embodiments, the binding moiety A is conjugated to a polynucleicacid molecule (B) in a site-specific manner. In some instances, thebinding moiety A is conjugated to a polynucleic acid molecule (B)through a lysine residue, a cysteine residue, at the 5′-terminus, at the3′-terminus, an unnatural amino acid, or an enzyme-modified orenzyme-catalyzed residue, via a site-specific manner. In some instances,the binding moiety A is conjugated to a polynucleic acid molecule (B)through a lysine residue (e.g., lysine residue present in the bindingmoiety A) via a site-specific manner. In some instances, the bindingmoiety A is conjugated to a polynucleic acid molecule (B) through acysteine residue (e.g., cysteine residue present in the binding moietyA) via a site-specific manner. In some instances, the binding moiety Ais conjugated to a polynucleic acid molecule (B) at the 5′-terminus viaa site-specific manner. In some instances, the binding moiety A isconjugated to a polynucleic acid molecule (B) at the 3′-terminus via asite-specific manner. In some instances, the binding moiety A isconjugated to a polynucleic acid molecule (B) through an unnatural aminoacid via a site-specific manner. In some instances, the binding moiety Ais conjugated to a polynucleic acid molecule (B) through anenzyme-modified or enzyme-catalyzed residue via a site-specific manner.

In some embodiments, one or more polynucleic acid molecule (B) isconjugated to a binding moiety A. In some instances, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 1 polynucleic acid molecule is conjugated to one binding moiety A.In some instances, about 2 polynucleic acid molecules are conjugated toone binding moiety A. In some instances, about 3 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 4 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 5 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 6 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 7 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 8 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 9 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 10 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 11 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 12 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 13 polynucleic acid molecules are conjugated to one binding moietyA. In some instances, about 14 polynucleic acid molecules are conjugatedto one binding moiety A. In some instances, about 15 polynucleic acidmolecules are conjugated to one binding moiety A. In some instances,about 16 polynucleic acid molecules are conjugated to one binding moietyA. In some cases, the one or more polynucleic acid molecules are thesame. In other cases, the one or more polynucleic acid molecules aredifferent.

In some embodiments, the number of polynucleic acid molecule (B)conjugated to a binding moiety A forms a ratio. In some instances, theratio is referred to as a DAR (drug-to-antibody) ratio, in which thedrug as referred to herein is the polynucleic acid molecule (B). In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,or greater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 1 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 2 or greater. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 3 or greater.In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 4 or greater. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is about 5 orgreater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 6 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 7 or greater. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 8 or greater.In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 9 or greater. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is about 10 orgreater. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 11 or greater. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 12 or greater.

In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 1. In some instances, the DARratio of the polynucleic acid molecule (B) to binding moiety A is about2. In some instances, the DAR ratio of the polynucleic acid molecule (B)to binding moiety A is about 3. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 4. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 5. In some instances, the DAR ratio of the polynucleicacid molecule (B) to binding moiety A is about 6. In some instances, theDAR ratio of the polynucleic acid molecule (B) to binding moiety A isabout 7. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is about 8. In some instances, the DARratio of the polynucleic acid molecule (B) to binding moiety A is about9. In some instances, the DAR ratio of the polynucleic acid molecule (B)to binding moiety A is about 10. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 11. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 12. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 13. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 14. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is about 15. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is about 16.

In some instances, the DAR ratio of the polynucleic acid molecule (B) tobinding moiety A is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16. In some instances, the DAR ratio of the polynucleic acid molecule(B) to binding moiety A is 1. In some instances, the DAR ratio of thepolynucleic acid molecule (B) to binding moiety A is 2. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is 4. In some instances, the DAR ratio of the polynucleic acidmolecule (B) to binding moiety A is 6. In some instances, the DAR ratioof the polynucleic acid molecule (B) to binding moiety A is 8. In someinstances, the DAR ratio of the polynucleic acid molecule (B) to bindingmoiety A is 12.

In some instances, a conjugate comprising polynucleic acid molecule (B)and binding moiety A has improved activity as compared to a conjugatecomprising polynucleic acid molecule (B) without a binding moiety A. Insome instances, improved activity results in enhanced biologicallyrelevant functions, e.g., improved stability, affinity, binding,functional activity, and efficacy in treatment or prevention of adisease state. In some instances, the disease state is a result of oneor more mutated exons of a gene. In some instances, the conjugatecomprising polynucleic acid molecule (B) and binding moiety A results inincreased exon skipping of the one or more mutated exons as compared tothe conjugate comprising polynucleic acid molecule (B) without a bindingmoiety A. In some instances, exon skipping is increased by at least orabout 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or morethan 95% in the conjugate comprising polynucleic acid molecule (B) andbinding moiety A as compared to the conjugate comprising polynucleicacid molecule (B) without a binding moiety A.

In some embodiments, an antibody or antigen binding fragment is furthermodified using conventional techniques known in the art, for example, byusing amino acid deletion, insertion, substitution, addition, and/or byrecombination and/or any other modification (e.g., posttranslational andchemical modifications, such as glycosylation and phosphorylation) knownin the art either alone or in combination. In some instances, themodification further comprises a modification for modulating interactionwith Fc receptors. In some instances, the one or more modificationsinclude those described in, for example, International Publication No.WO97/34631, which discloses amino acid residues involved in theinteraction between the Fc domain and the FcRn receptor. Methods forintroducing such modifications in the nucleic acid sequence underlyingthe amino acid sequence of an antibody or antigen binding fragment iswell known to the person skilled in the art.

In some instances, an antigen binding fragment further encompasses itsderivatives and includes polypeptide sequences containing at least oneCDR.

In some instances, the term “single-chain” as used herein means that thefirst and second domains of a bi-specific single chain construct arecovalently linked, preferably in the form of a co-linear amino acidsequence encodable by a single nucleic acid molecule.

In some instances, a bispecific single chain antibody construct relatesto a construct comprising two antibody derived binding domains. In suchembodiments, bi-specific single chain antibody construct is tandembi-scFv or diabody. In some instances, a scFv contains a VH and VLdomain connected by a linker peptide. In some instances, linkers are ofa length and sequence sufficient to ensure that each of the first andsecond domains can, independently from one another, retain theirdifferential binding specificities.

In some embodiments, binding to or interacting with as used hereindefines a binding/interaction of at least two antigen-interaction-siteswith each other. In some instances, antigen-interaction-site defines amotif of a polypeptide that shows the capacity of specific interactionwith a specific antigen or a specific group of antigens. In some cases,the binding/interaction is also understood to define a specificrecognition. In such cases, specific recognition refers to that theantibody or its antigen binding fragment is capable of specificallyinteracting with and/or binding to at least two amino acids of each of atarget molecule. For example, specific recognition relates to thespecificity of the antibody molecule, or to its ability to discriminatebetween the specific regions of a target molecule. In additionalinstances, the specific interaction of the antigen-interaction-site withits specific antigen results in an initiation of a signal, e.g. due tothe induction of a change of the conformation of the antigen, anoligomerization of the antigen, etc. In further embodiments, the bindingis exemplified by the specificity of a “key-lock-principle”. Thus insome instances, specific motifs in the amino acid sequence of theantigen-interaction-site and the antigen bind to each other as a resultof their primary, secondary or tertiary structure as well as the resultof secondary modifications of said structure. In such cases, thespecific interaction of the antigen-interaction-site with its specificantigen results as well in a simple binding of the site to the antigen.

In some instances, specific interaction further refers to a reducedcross-reactivity of the antibody or antigen binding fragment or areduced off-target effect. For example, the antibody or antigen bindingfragment that bind to the polypeptide/protein of interest but do not ordo not essentially bind to any of the other polypeptides are consideredas specific for the polypeptide/protein of interest. Examples for thespecific interaction of an antigen-interaction-site with a specificantigen comprise the specificity of a ligand for its receptor, forexample, the interaction of an antigenic determinant (epitope) with theantigenic binding site of an antibody.

Additional Binding Moieties

In some embodiments, the binding moiety is a plasma protein. In someinstances, the plasma protein comprises albumin. In some instances, thebinding moiety A is albumin. In some instances, albumin is conjugated byone or more of a conjugation chemistry described herein to a polynucleicacid molecule. In some instances, albumin is conjugated by nativeligation chemistry to a polynucleic acid molecule. In some instances,albumin is conjugated by lysine conjugation to a polynucleic acidmolecule.

In some instances, the binding moiety is a steroid. Exemplary steroidsinclude cholesterol, phospholipids, di- and triacylglycerols, fattyacids, hydrocarbons that are saturated, unsaturated, comprisesubstitutions, or combinations thereof. In some instances, the steroidis cholesterol. In some instances, the binding moiety is cholesterol. Insome instances, cholesterol is conjugated by one or more of aconjugation chemistry described herein to a polynucleic acid molecule.In some instances, cholesterol is conjugated by native ligationchemistry to a polynucleic acid molecule. In some instances, cholesterolis conjugated by lysine conjugation to a polynucleic acid molecule.

In some instances, the binding moiety is a polymer, including but notlimited to polynucleic acid molecule aptamers that bind to specificsurface markers on cells. In this instance the binding moiety is apolynucleic acid that does not hybridize to a target gene or mRNA, butinstead is capable of selectively binding to a cell surface markersimilarly to an antibody binding to its specific epitope of a cellsurface marker.

In some cases, the binding moiety is a peptide. In some cases, thepeptide comprises between about 1 and about 3 kDa. In some cases, thepeptide comprises between about 1.2 and about 2.8 kDa, about 1.5 andabout 2.5 kDa, or about 1.5 and about 2 kDa. In some instances, thepeptide is a bicyclic peptide. In some cases, the bicyclic peptide is aconstrained bicyclic peptide. In some instances, the binding moiety is abicyclic peptide (e.g., bicycles from Bicycle Therapeutics).

In additional cases, the binding moiety is a small molecule. In someinstances, the small molecule is an antibody-recruiting small molecule.In some cases, the antibody-recruiting small molecule comprises atarget-binding terminus and an antibody-binding terminus, in which thetarget-binding terminus is capable of recognizing and interacting with acell surface receptor. For example, in some instances, thetarget-binding terminus comprising a glutamate urea compound enablesinteraction with PSMA, thereby, enhances an antibody interaction with acell that expresses PSMA. In some instances, a binding moiety is a smallmolecule described in Zhang et al., “A remote arene-binding site onprostate specific membrane antigen revealed by antibody-recruiting smallmolecules,” J Am Chem Soc. 132(36): 12711-12716 (2010); or McEnaney, etal., “Antibody-recruiting molecules: an emerging paradigm for engagingimmune function in treating human disease,” ACS Chem Biol. 7(7):1139-1151 (2012).

Production of Antibodies or Antigen Binding Fragment Thereof

In some embodiments, polypeptides described herein (e.g., antibodies andantigen binding fragments) are produced using any method known in theart to be useful for the synthesis of polypeptides (e.g., antibodies),in particular, by chemical synthesis or by recombinant expression, andare preferably produced by recombinant expression techniques.

In some instances, an antibody or antigen binding fragment thereof isexpressed recombinantly, and the nucleic acid encoding the antibody orantigen binding fragment is assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., 1994.BioTechniques 17:242), which involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligation of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a nucleic acid molecule encoding an antibody isoptionally generated from a suitable source (e.g., an antibody cDNAlibrary, or cDNA library generated from any tissue or cells expressingthe immunoglobulin) by PCR amplification using synthetic primershybridizable to the 3′ and 5′ ends of the sequence or by cloning usingan oligonucleotide probe specific for the particular gene sequence.

In some instances, an antibody or its antigen binding is optionallygenerated by immunizing an animal, such as a rabbit, to generatepolyclonal antibodies or, more preferably, by generating monoclonalantibodies, e.g., as described by Kohler and Milstein (1975. Nature256:495-497) or, as described by Kozbor et al. (1983. Immunology Today4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, Inc., pp. 77-96). Alternatively, a clone encoding at leastthe Fab portion of the antibody is optionally obtained by screening Fabexpression libraries (e.g., as described in Huse et al., 1989. Science246:1275-1281) for clones of Fab fragments that bind the specificantigen or by screening antibody libraries (See, e.g., Clackson et al.,1991. Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA94:4937).

In some embodiments, techniques developed for the production of“chimeric antibodies” (Morrison et al., 1984. Proc. Natl. Acad. Sci.81:851-855; Neuberger et al., 1984. Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity are used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine monoclonal antibody and a human immunoglobulinconstant region, e.g., humanized antibodies.

In some embodiments, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988. Science242:423-42; Huston et al., 1988. Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989. Nature 334:544-54) are adapted toproduce single chain antibodies. Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide. Techniquesfor the assembly of functional Fv fragments in E. coli are alsooptionally used (Skerra et al., 1988. Science 242:1038-1041).

In some embodiments, an expression vector comprising the nucleotidesequence of an antibody or the nucleotide sequence of an antibody istransferred to a host cell by conventional techniques (e.g.,electroporation, liposomal transfection, and calcium phosphateprecipitation), and the transfected cells are then cultured byconventional techniques to produce the antibody. In specificembodiments, the expression of the antibody is regulated by aconstitutive, an inducible or a tissue, specific promoter.

In some embodiments, a variety of host-expression vector systems isutilized to express an antibody or its antigen binding fragmentdescribed herein. Such host-expression systems represent vehicles bywhich the coding sequences of the antibody is produced and subsequentlypurified, but also represent cells that are, when transformed ortransfected with the appropriate nucleotide coding sequences, express anantibody or its antigen binding fragment in situ. These include, but arenot limited to, microorganisms such as bacteria (e.g. E. coli and B.subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA orcosmid DNA expression vectors containing an antibody or its antigenbinding fragment coding sequences; yeast (e.g., Saccharomyces Pichia)transformed with recombinant yeast expression vectors containing anantibody or its antigen binding fragment coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing an antibody or its antigen binding fragmentcoding sequences; plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus (CaMV) and tobaccomosaic virus (TMV)) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing an antibody or its antigen bindingfragment coding sequences; or mammalian cell systems (e.g., COS, CHO,BH, 293, 293T, 3T3 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g. the adenoviruslate promoter; the vaccinia virus 7.5K promoter).

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. In some instances, cell lines that stablyexpress an antibody are optionally engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellsare transformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells are thenallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci that in turnare cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express the antibody or its antigenbinding fragments.

In some instances, a number of selection systems are used, including butnot limited to the herpes simplex virus thymidine kinase (Wigler et al.,1977. Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, 192. Proc. Natl. Acad. Sci. USA 48:202), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genesare employed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance are used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980. Proc. Natl. Acad Sci. USA 77:357; O'Hare et al., 1981,Proc. Natl. Acad Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;Tolstoshev, 1993. Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan,1993. Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev.Biochem. 62:191-217; May, 1993. TIB TECH 11(5):155-215) and hygro, whichconfers resistance to hygromycin (Santerre et al., 1984, Gene 30:147).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds., 1993. CurrentProtocols m Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990.Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY;and in Chapters 12 and 13, Dracopoli et al. (eds), 1994. CurrentProtocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin etal., 1981, J. Mol. Biol. 150:1).

In some instances, the expression levels of an antibody are increased byvector amplification (for a review, see Bebbington and Hentschel, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, NewYork, 1987)). When a marker in the vector system expressing an antibodyis amplifiable, an increase in the level of inhibitor present in cultureof host cell will increase the number of copies of the marker gene.Since the amplified region is associated with the nucleotide sequence ofthe antibody, production of the antibody will also increase (Crouse etal., 1983, Mol. Cell Biol. 3:257).

In some instances, any method known in the art for purification oranalysis of an antibody or antibody conjugates is used, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Exemplarychromatography methods included, but are not limited to, strong anionexchange chromatography, hydrophobic interaction chromatography, sizeexclusion chromatography, and fast protein liquid chromatography.

Conjugation Chemistry

In some embodiments, a polynucleic acid molecule B is conjugated to abinding moiety. In some embodiments, a polynucleic acid molecule B isconjugated to a binding moiety in a formula A-X-B (X is a linkerconjugating A and B). In some instances, the binding moiety comprisesamino acids, peptides, polypeptides, proteins, antibodies, antigens,toxins, hormones, lipids, nucleotides, nucleosides, sugars,carbohydrates, polymers such as polyethylene glycol and polypropyleneglycol, as well as analogs or derivatives of all of these classes ofsubstances. Additional examples of binding moiety also include steroids,such as cholesterol, phospholipids, di- and triacylglycerols, fattyacids, hydrocarbons (e.g., saturated, unsaturated, or containssubstitutions), enzyme substrates, biotin, digoxigenin, andpolysaccharides. In some instances, the binding moiety is an antibody orantigen binding fragment thereof. In some instances, the polynucleicacid molecule is further conjugated to a polymer, and optionally anendosomolytic moiety.

In some embodiments, the polynucleic acid molecule is conjugated to thebinding moiety by a chemical ligation process. In some instances, thepolynucleic acid molecule is conjugated to the binding moiety by anative ligation. In some instances, the conjugation is as described in:Dawson, et al. “Synthesis of proteins by native chemical ligation.”Science 1994, 266, 776-779; Dawson, et al. “Modulation of Reactivity inNative Chemical Ligation through the Use of Thiol Additives,” J. Am.Chem. Soc. 1997, 119, 4325-4329; Hackeng, et al. “Protein synthesis bynative chemical ligation: Expanded scope by using straightforwardmethodology.,” Proc. Natl. Acad Sci. USA 1999, 96, 10068-10073; or Wu,et al. “Building complex glycopeptides: Development of a cysteine-freenative chemical ligation protocol,” Angew. Chem. Int. Ed. 2006, 45,4116-125. In some instances, the conjugation is as described in U.S.Pat. No. 8,936,910. In some embodiments, the polynucleic acid moleculeis conjugated to the binding moiety either site-specifically ornon-specifically via native ligation chemistry.

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing a “traceless”coupling technology (Philochem). In some instances, the “traceless”coupling technology utilizes an N-terminal 1,2-aminothiol group on thebinding moiety which is then conjugate with a polynucleic acid moleculecontaining an aldehyde group. (see Casi et al., “Site-specific tracelesscoupling of potent cytotoxic drugs to recombinant antibodies forpharmacodelivery,” JACS 134(13): 5887-5892 (2012))

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing an unnatural aminoacid incorporated into the binding moiety. In some instances, theunnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In someinstances, the keto group of pAcPhe is selectively coupled to analkoxy-amine derivatived conjugating moiety to form an oxime bond. (seeAxup et al., “Synthesis of site-specific antibody-drug conjugates usingunnatural amino acids,” PNAS 109(40): 16101-16106 (2012)).

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a site-directed method utilizing an enzyme-catalyzedprocess. In some instances, the site-directed method utilizes SMARTag™technology (Catalent, Inc.). In some instances, the SMARTag™ technologycomprises generation of a formylglycine (FGly) residue from cysteine byformylglycine-generating enzyme (FGE) through an oxidation process underthe presence of an aldehyde tag and the subsequent conjugation of FGlyto an alkylhydraine-functionalized polynucleic acid molecule viahydrazino-Pictet-Spengler (HIPS) ligation. (see Wu et al.,“Site-specific chemical modification of recombinant proteins produced inmammalian cells by using the genetically encoded aldehyde tag,” PNAS106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet-Spengler ligationfor protein chemical modification,” PNAS 110(1): 46-51 (2013))

In some instances, the enzyme-catalyzed process comprises microbialtransglutaminase (mTG). In some cases, the polynucleic acid molecule isconjugated to the binding moiety utilizing a microbialtransglutaminase-catalyzed process. In some instances, mTG catalyzes theformation of a covalent bond between the amide side chain of a glutaminewithin the recognition sequence and a primary amine of a functionalizedpolynucleic acid molecule. In some instances, mTG is produced fromStreptomyces mobarensis. (see Strop et al., “Location matters: site ofconjugation modulates stability and pharmacokinetics of antibody drugconjugates,” Chemistry and Biology 20(2) 161-167 (2013))

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a method as described in PCT Publication No.WO2014/140317, which utilizes a sequence-specific transpeptidase.

In some instances, the polynucleic acid molecule is conjugated to thebinding moiety by a method as described in U.S. Patent Publication Nos.2015/0105539 and 2015/0105540.

Polymer Conjugating Moiety

In some embodiments, a polymer moiety C is further conjugated to apolynucleic acid molecule described herein, a binding moiety describedherein, or in combinations thereof. In some instances, a polymer moietyC is conjugated a polynucleic acid molecule in a formula A-X₁-B-X₂—C(X₁,X₂ as two linkers conjugating A and B, B and C, respectively). In somecases, a polymer moiety C is conjugated to a binding moiety. In othercases, a polymer moiety C is conjugated to a polynucleic acidmolecule-binding moiety molecule. In additional cases, a polymer moietyC is conjugated, as illustrated supra.

In some instances, the polymer moiety C is a natural or syntheticpolymer, consisting of long chains of branched or unbranched monomers,and/or cross-linked network of monomers in two or three dimensions. Insome instances, the polymer moiety C includes a polysaccharide, lignin,rubber, or polyalkylen oxide (e.g., polyethylene glycol). In someinstances, the at least one polymer moiety C includes, but is notlimited to, alpha-, omega-dihydroxylpolyethyleneglycol, biodegradablelactone-based polymer, e.g. polyacrylic acid, polylactide acid (PLA),poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin,polyamide, polycyanoacrylate, polyimide, polyethylene terephthalate(also known as poly(ethylene terephthalate), PET, PETG, or PETE),polytetramethylene glycol (PTG), or polyurethane as well as mixturesthereof. As used herein, a mixture refers to the use of differentpolymers within the same compound as well as in reference to blockcopolymers. In some cases, block copolymers are polymers wherein atleast one section of a polymer is build up from monomers of anotherpolymer. In some instances, the polymer moiety C comprises polyalkyleneoxide. In some instances, the polymer moiety C comprises PEG. In someinstances, the polymer moiety C comprises polyethylene imide (PEI) orhydroxy ethyl starch (HES).

In some instances, C is a PEG moiety. In some instances, the PEG moietyis conjugated at the 5′ terminus of the polynucleic acid molecule whilethe binding moiety is conjugated at the 3′ terminus of the polynucleicacid molecule. In some instances, the PEG moiety is conjugated at the 3′terminus of the polynucleic acid molecule while the binding moiety isconjugated at the 5′ terminus of the polynucleic acid molecule. In someinstances, the PEG moiety is conjugated to an internal site of thepolynucleic acid molecule. In some instances, the PEG moiety, thebinding moiety, or a combination thereof, are conjugated to an internalsite of the polynucleic acid molecule. In some instances, theconjugation is a direct conjugation. In some instances, the conjugationis via native ligation.

In some embodiments, the polyalkylene oxide (e.g., PEG) is apolydisperse or monodisperse compound. In some instances, polydispersematerial comprises disperse distribution of different molecular weightof the material, characterized by mean weight (weight average) size anddispersity. In some instances, the monodisperse PEG comprises one sizeof molecules. In some embodiments, C is poly- or monodispersedpolyalkylene oxide (e.g., PEG) and the indicated molecular weightrepresents an average of the molecular weight of the polyalkylene oxide,e.g., PEG, molecules.

In some embodiments, the molecular weight of the polyalkylene oxide(e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750,4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000,10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.

In some embodiments, C is polyalkylene oxide (e.g., PEG) and has amolecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100,2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500,3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500,8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100.000Da. In some embodiments, C is PEG and has a molecular weight of about200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500,2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500,4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000,20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In someinstances, the molecular weight of C is about 200 Da. In some instances,the molecular weight of C is about 300 Da. In some instances, themolecular weight of C is about 400 Da. In some instances, the molecularweight of C is about 500 Da. In some instances, the molecular weight ofC is about 600 Da. In some instances, the molecular weight of C is about700 Da. In some instances, the molecular weight of C is about 800 Da. Insome instances, the molecular weight of C is about 900 Da. In someinstances, the molecular weight of C is about 1000 Da. In someinstances, the molecular weight of C is about 1100 Da. In someinstances, the molecular weight of C is about 1200 Da. In someinstances, the molecular weight of C is about 1300 Da. In someinstances, the molecular weight of C is about 1400 Da. In someinstances, the molecular weight of C is about 1450 Da. In someinstances, the molecular weight of C is about 1500 Da. In someinstances, the molecular weight of C is about 1600 Da. In someinstances, the molecular weight of C is about 1700 Da. In someinstances, the molecular weight of C is about 1800 Da. In someinstances, the molecular weight of C is about 1900 Da. In someinstances, the molecular weight of C is about 2000 Da. In someinstances, the molecular weight of C is about 2100 Da. In someinstances, the molecular weight of C is about 2200 Da. In someinstances, the molecular weight of C is about 2300 Da. In someinstances, the molecular weight of C is about 2400 Da. In someinstances, the molecular weight of C is about 2500 Da. In someinstances, the molecular weight of C is about 2600 Da. In someinstances, the molecular weight of C is about 2700 Da. In someinstances, the molecular weight of C is about 2800 Da. In someinstances, the molecular weight of C is about 2900 Da. In someinstances, the molecular weight of C is about 3000 Da. In someinstances, the molecular weight of C is about 3250 Da. In someinstances, the molecular weight of C is about 3350 Da. In someinstances, the molecular weight of C is about 3500 Da. In someinstances, the molecular weight of C is about 3750 Da. In someinstances, the molecular weight of C is about 4000 Da. In someinstances, the molecular weight of C is about 4250 Da. In someinstances, the molecular weight of C is about 4500 Da. In someinstances, the molecular weight of C is about 4600 Da. In someinstances, the molecular weight of C is about 4750 Da. In someinstances, the molecular weight of C is about 5000 Da. In someinstances, the molecular weight of C is about 5500 Da. In someinstances, the molecular weight of C is about 6000 Da. In someinstances, the molecular weight of C is about 6500 Da. In someinstances, the molecular weight of C is about 7000 Da. In someinstances, the molecular weight of C is about 7500 Da. In someinstances, the molecular weight of C is about 8000 Da. In someinstances, the molecular weight of C is about 10,000 Da. In someinstances, the molecular weight of C is about 12,000 Da. In someinstances, the molecular weight of C is about 20,000 Da. In someinstances, the molecular weight of C is about 35,000 Da. In someinstances, the molecular weight of C is about 40,000 Da. In someinstances, the molecular weight of C is about 50,000 Da. In someinstances, the molecular weight of C is about 60,000 Da. In someinstances, the molecular weight of C is about 100,000 Da.

In some embodiments, the polyalkylene oxide (e.g., PEG) comprisesdiscrete ethylene oxide units (e.g., four to about 48 ethylene oxideunits). In some instances, the polyalkylene oxide comprising thediscrete ethylene oxide units is a linear chain. In other cases, thepolyalkylene oxide comprising the discrete ethylene oxide units is abranched chain.

In some instances, the polymer moiety C is a polyalkylene oxide (e.g.,PEG) comprising discrete ethylene oxide units. In some cases, thepolymer moiety C comprises between about 4 and about 48 ethylene oxideunits. In some cases, the polymer moiety C comprises about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, about 24, about 25, about 26, about27, about 28, about 29, about 30, about 31, about 32, about 33, about34, about 35, about 36, about 37, about 38, about 39, about 40, about41, about 42, about 43, about 44, about 45, about 46, about 47, or about48 ethylene oxide units.

In some instances, the polymer moiety C is a discrete PEG comprising,e.g., between about 4 and about 48 ethylene oxide units. In some cases,the polymer moiety C is a discrete PEG comprising, e.g., about 4, about5, about 6, about 7, about 8, about 9, about 10, about 11, about 12,about 13, about 14, about 15, about 16, about 17, about 18, about 19,about 20, about 21, about 22, about 23, about 24, about 25, about 26,about 27, about 28, about 29, about 30, about 31, about 32, about 33,about 34, about 35, about 36, about 37, about 38, about 39, about 40,about 41, about 42, about 43, about 44, about 45, about 46, about 47, orabout 48 ethylene oxide units. In some cases, the polymer moiety C is adiscrete PEG comprising, e.g., about 4 ethylene oxide units. In somecases, the polymer moiety C is a discrete PEG comprising, e.g., about 5ethylene oxide units. In some cases, the polymer moiety C is a discretePEG comprising, e.g., about 6 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 7 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 8 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 9 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 10 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 11 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 12 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 13 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 14 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 15 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising. e.g., about 16 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 17 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 18 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 19 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 20 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 21 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 22 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 23 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 24 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising. e.g., about 25 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 26 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g. about 27 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 28 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 29 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 30 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 31 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 32 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 33 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 34 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 35 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 36 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 37 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 38 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 39 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising. e.g., about 40 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 41 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 42 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 43 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 44 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 45 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 46 ethylene oxide units. In some cases, thepolymer moiety C is a discrete PEG comprising, e.g., about 47 ethyleneoxide units. In some cases, the polymer moiety C is a discrete PEGcomprising, e.g., about 48 ethylene oxide units.

In some cases, the polymer moiety C is dPEG® (Quanta Biodesign Ltd).

In some embodiments, the polymer moiety C comprises a cationic mucicacid-based polymer (cMAP). In some instances, cMAP comprises one or moresubunit of at least one repeating subunit, and the subunit structure isrepresented as Formula (V):

-   -   wherein m is independently at each occurrence 1, 2, 3, 4, 5, 6,        7, 8, 9, or 10, preferably 4-6 or 5; and n is independently at        each occurrence 1, 2, 3, 4, or 5. In some embodiments, m and n        are, for example, about 10.

In some instances, cMAP is further conjugated to a PEG moiety,generating a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, ora cMAP-PEG-cMAP triblock polymer. In some instances, the PEG moiety isin a range of from about 500 Da to about 50,000 Da. In some instances,the PEG moiety is in a range of from about 500 Da to about 1000 Da,greater than 1000 Da to about 5000 Da, greater than 5000 Da to about10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000Da to about 50,000 Da, or any combination of two or more of theseranges.

In some instances, the polymer moiety C is cMAP-PEG copolymer, anmPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. Insome cases, the polymer moiety C is cMAP-PEG copolymer. In other cases,the polymer moiety C is an mPEG-cMAP-PEGm triblock polymer. Inadditional cases, the polymer moiety C is a cMAP-PEG-cMAP triblockpolymer.

In some embodiments, the polymer moiety C is conjugated to thepolynucleic acid molecule, the binding moiety, and optionally to theendosomolytic moiety as illustrated supra.

Endosomolytic or Cell Membrane Penetration Moiety

In some embodiments, a molecule of Formula (I): A-X₁-B-X₂-C, furthercomprises an additional conjugating moiety. In some instances, theadditional conjugating moiety is an endosomolytic moiety and/or a cellmembrane penetration moiety. In some cases, the endosomolytic moiety isa cellular compartmental release component, such as a compound capableof releasing from any of the cellular compartments known in the art,such as the endosome, lysosome, endoplasmic reticulum (ER), Golgiapparatus, microtubule, peroxisome, or other vesicular bodies with thecell. In some cases, the endosomolytic moiety comprises an endosomolyticpolypeptide, an endosomolytic polymer, an endosomolytic lipid, or anendosomolytic small molecule. In some cases, the endosomolytic moietycomprises an endosomolytic polypeptide. In other cases, theendosomolytic moiety comprises an endosomolytic polymer. In some cases,the cell membrane penetration moiety comprises a cell penetratingpeptide (CPP). In other cases, the cell membrane penetration moietycomprises a cell penetrating lipid. In other cases, the cell membranepenetration moiety comprises a cell penetrating small molecule.

Endosomolytic and Cell Membrane Penetration Polypeptides

In some embodiments, a molecule of Formula (I): A-X₁-B-X₂-C, is furtherconjugated with an endosomolytic polypeptide. In some cases, theendosomolytic polypeptide is a pH-dependent membrane active peptide. Insome cases, the endosomolytic polypeptide is an amphipathic polypeptide.In additional cases, the endosomolytic polypeptide is a peptidomimetic.In some instances, the endosomolytic polypeptide comprises INF,melittin, meucin, or their respective derivatives thereof. In someinstances, the endosomolytic polypeptide comprises INF or itsderivatives thereof. In other cases, the endosomolytic polypeptidecomprises melittin or its derivatives thereof. In additional cases, theendosomolytic polypeptide comprises meucin or its derivatives thereof.

In some instances, INF7 is a 24 residue polypeptide those sequencecomprises CGIFGEIEELIEEGLENLIDWGNA (SEQ ID NO: 331), orGLFEAIEGFIENGWEGMIDGWYGC (SEQ ID NO: 332). In some instances, INF7 orits derivatives comprise a sequence of: GLFEAIEGFIENGWEGMIWDYGSGSCG (SEQID NO: 333), GLFEAIEGFIENGWEGMIDG WYG-(PEG)6-NH2 (SEQ ID NO: 334), orGLFEAIEGFIENGWEGMIWDYG-SGSC-K(GaJNAc)2 (SEQ ID NO: 335).

In some cases, melittin is a 26 residue polypeptide those sequencecomprises CLIGAILKVLATGLPTLISWIKNKRKQ (SEQ ID NO. 336), orGIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 337) In some instances, melittincomprises a polypeptide sequence as described in U.S. Pat. No.8,501,930.

In some instances, meucin is an antimicrobial peptide (AMP) derived fromthe venom gland of the scorpion Mesobuthus eupeus. In some instances,meucin comprises of meucin-13 those sequence comprises IFGAIAGLLKNIF-NH₂(SEQ ID NO: 338) and meucin-18 those sequence comprisesFFGHLFKLATKIIPSLFQ (SEQ ID NO: 339).

In some instances, the endosomolytic polypeptide comprises a polypeptidein which its sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%sequence identity to INF7 or its derivatives thereof, melittin or itsderivatives thereof, or meucin or its derivatives thereof. In someinstances, the endosomolytic moiety comprises INF7 or its derivativesthereof, melittin or its derivatives thereof, or meucin or itsderivatives thereof.

In some instances, the endosomolytic moiety is INF7 or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 331-335. In somecases, the endosomolytic moiety comprises a polypeptide having at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90°,%, 95%, 96%, 97%, 98%, 99%,or 100% sequence identity to SEQ ID NO: 331. In some cases, theendosomolytic moiety comprises a polypeptide having at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 332-335. In some cases, theendosomolytic moiety comprises SEQ ID NO: 331. In some cases, theendosomolytic moiety comprises SEQ ID NO: 332-335. In some cases, theendosomolytic moiety consists of SEQ ID NO: 331. In some cases, theendosomolytic moiety consists of SEQ ID NO: 332-335.

In some instances, the endosomolytic moiety is melittin or itsderivatives thereof. In some cases, the endosomolytic moiety comprises apolypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 336 or337. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 336. In somecases, the endosomolytic moiety comprises a polypeptide having at least50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NO: 337. In some cases, theendosomolytic moiety comprises SEQ ID NO: 286. In some cases, theendosomolytic moiety comprises SEQ ID NO: 337. In some cases, theendosomolytic moiety consists of SEQ ID NO: 336. In some cases, theendosomolytic moiety consists of SEQ ID NO: 337.

In some instances, the endosomolytic moiety is meucin or its derivativesthereof. In some cases, the endosomolytic moiety comprises a polypeptidehaving at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 338 or 339. Insome cases, the endosomolytic moiety, comprises a polypeptide having atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% sequence identity to SEQ ID NO: 338. In some cases, theendosomolytic moiety comprises a polypeptide having at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to SEQ ID NO: 339. In some cases, the endosomolyticmoiety comprises SEQ ID NO: 338. In some cases, the endosomolytic moietycomprises SEQ ID NO: 339. In some cases, the endosomolytic moietyconsists of SEQ ID NO: 338. In some cases, the endosomolytic moietyconsists of SEQ ID NO: 339.

In some instances, the endosomolytic moiety comprises a sequence asillustrated in Table 8

TABLE 8 SEQ ID NAME ORIGIN AMINO ACID SEQUENCE NO: TYPE Pep-1NLS from Simian Virus KETWWETWWTEWSQPKKKRKV 340 Primary40 large antigen and amphipathic Reverse transcriptase of HIV pVECVE-cadherin LLIILRRRRIRKQAHAHSK 341 Primary amphipathic VT5Synthetic peptide DPKGDPKGVTVTVTVTVTGKGDP 342 β-sheet KPD amphipathicC105Y 1-antitrypsin CSIPPEVKFNKPFVYLI 343 — TransportanGalanin and mastoparan GWTLNSAGYLLGKINLKALAALA 344 Primary KKILamphipathic TP10 Galanin and mastoparan AGYLLGKINLKALAALAKKIL 345Primary amphipathic MPG A hydrophobic domain GALFLGFLGAAGSTMGA 346β-sheet from the fusion amphipathic sequence of HIV gp41and NLS of SV40 T antigen gH625 Glycoprotein gH of HGLASTLTRWAHYNALIRAF347 Secondary HSV type I amphipathic α-helical CADY PPTG1 peptideGLWRALWRLLRSLWRLLWRA 348 Secondary amphipathic α-helical GALASynthetic peptide WEAALAEALAEALAEHLAEALAE 349 Secondary ALEALAAamphipathic α-helical INF Influenza HA2 fusion GLFEAIEGFIENGWEGMIDGWYGC350 Secondary peptide amphipathic α-helical/ pH- dependent membraneactive peptide HA2E5- Influenza HA2 subunit GLFGAIAGFIENGWEGMIDGWYG 351Secondary TAT of influenza virus X31 amphipathic strain fusion peptideα-helical/ pH- dependent membrane active peptide HA2-Influenza HA2 subunit GLFGAIAGFIENGWEGMIDGRQIKI 352 pH- penetratinof influenza virus X31 WFQNRRMKW dependent strain fusion peptideKK-amide membrane active peptide HA-K4 Influenza HA2 subunitGLFGAIAGFIENGWEGMIDG- 353 pH- of influenza virus X31 SSKKKK dependentstrain fusion peptide membrane active peptide HA2E4Influenza HA2 subunit GLFEAIAGFIENGWEGMIDGGGYC 354 pH-of influenza virus X31 dependent strain fusion peptide membrane activepeptide H5WYG HA2 analogue GLFHAIAHFIHGGWH 355 pH- GLIHGWYG dependentmembrane active peptide GALA- INF3 fusion peptideGLFEAIEGFIENGWEGLAEALAEAL 356 pH- INF3- EALAA- dependent (PEG)6-NH(PEG)6-NH2 membrane active peptide CM18- Cecropin-A-Melittin₂₋₁₂KWKLFKKIGAVLKVLTTG- 357 pH- TAT11 (CM₁₈) fusion peptide YGRKKRRQRRRdependent membrane active peptide

In some cases, the endosomolytic moiety comprises a Bak BH3 polypeptidewhich induces apoptosis through antagonization of suppressor targetssuch as Bcl-2 and/or Bcl-x_(L). In some instances, the endosomolyticmoiety comprises a Bak BH3 polypeptide described in Albarran, et al.,“Efficient intracellular delivery of a pro-apoptotic peptide with apH-responsive carrier,” Reactive & Functional Polymers 71: 261-265(2011).

In some instances, the endosomolytic moiety comprises a polypeptide(e.g., a cell-penetrating polypeptide) as described in PCT PublicationNos. WO20131166155 or WO20151069587.

Endosomolytic Lipids

In some embodiments, the endosomolytic moiety is a lipid (e.g., afusogenic lipid). In some embodiments, a molecule of Formula (I):A-X₁-B-X₂-C, is further conjugated with an endosomolytic lipid (e.g.,fusogenic lipid). Exemplary fusogenic lipids include1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine(POPE), palmitoyloleoylphosphatidvlcholine (POPC),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin),N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine(DLin-k-DMA) andN-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine(XTC).

In some instances, an endosomolytic moiety is a lipid (e.g., a fusogeniclipid) described in PCT Publication No. WO09/126,933.

Endosomolytic Small Molecules

In some embodiments, the endosomolytic moiety is a small molecule. Insome embodiments, a molecule of Formula (I). A-X₁-B-X₂-C, is furtherconjugated with an endosomolytic small molecule. Exemplary smallmolecules suitable as endosomolytic moieties include, but are notlimited to, quinine, chloroquine, hydroxychloroquines, amodiaquins(camoquines), amopyroquines, primaquines, mefloquines, nivaquines,halofantrines, quinone imines, or a combination thereof. In someinstances, quinoline endosomolytic moieties include, but are not limitedto, 7-chloro-4-(4-diethylamino-1-methylbutyl-amino)quinoline(chloroquine);7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl-amino)quinoline(hydroxychloroquine);7-fluoro-4-(4-diethylamino-1-methylbutyl-amino)quinoline:4-(4-diethylamino-1-methylbutylamino) quinoline;7-hydroxy-4-(4-diethyl-amino-1-methylbutylamino)quinoline;7-chloro-4-(4-diethylamino-1-butylamino)quinoline(desmethylchloroquine);7-fluoro-4-(4-diethylamino-1-butylamino)quinoline);4-(4-diethyl-amino-1-butylamino)quinoline;7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethyl-amino-1-butylamino)quinoline;4-(1-carboxy-4-diethylamino-1-butylamino) quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-diethyl-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;4-(4-ethyl-(2-hydroxy-ethyl)-amino-1-methylbutylamino-)quinoline;7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;hydroxychloroquine phosphate;7-chloro-4-(4-ethyl-(2-hydroxyethyl-1)-amino-1-butylamino)quinoline(desmethylhydroxychloroquine);7-fluoro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino) quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline;7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-fluoro-4-(1-carboxy-4-ethyl-(2-hydroethyl)-amino-1-methylbutylamino)quinoline;4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)quinoline;8-[(4-aminopentyl)amino]-6-methoxydihydrochloride quinoline;I-acetyl-1,2,3,4-tetrahydroquinoline;8-[(4-aminopentyl)amino]-6-methoxyquinoline dihydrochloride;1-butyryl-1,2,3,4-tetrahydroquinoline;3-chloro-4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,4-[(4-diethyl-amino)-1-methylbutyl-aminol-6-methoxyquinoline;3-fluoro-4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline,4-[(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;4-(4-hydroxy-alpha,alpha′-bis(2-methyl-1-pyrrolidinyl)-2,5-xylidinoquinoline;4-|(4-diethylamino)-1-methylbutyl-amino]-6-methoxyquinoline;3,4-dihydro-1-(2H)-quinolinecarboxaldehyde; 1,1′-pentamethylenediquinoleinium diiodide; 8-quinolinol sulfate and amino, aldehyde,carboxylic, hydroxyl, halogen, keto, sulthydryl and vinyl derivatives oranalogs thereof. In some instances, an endosomolytic moiety is a smallmolecule described in Naisbitt et al (1997, J Pharmacol Exp Therapy280:884-893) and in U.S. Pat. No. 5,736,557.

Cell Penetrating Polypeptide (CPP)

In some embodiments, cell penetrating polypeptide comprises positivelycharged short peptides with 5-30 amino acids. In some embodiments, cellpenetrating polypeptide comprises arginine or lysine rich amino acidsequences. In some embodiments, cell penetrating polypeptide includesany polypeptide or combination thereof listed in Table 9

TABLE 9 Peptide Sequence SEQ ID NO Antennapedia Penetratin (43-58)RQIKIWFQNRRMKWKK 358 HIV-1 TAT protein (48-60) GRKKRRQRRRPPQ 359pVEC Cadherin (615-632) LLIILRRRIRKQAHAHSK 360Transportan Galanine/Mastoparan GWTLNSAGYLLGKINLKALAALAKKIL 361MPG HIV-gp41/SV40 T-antigen GALFLGFLGAAGSTMGAWSQPKKKRKV 362Pep-1 HIV-reverse KETWWETWWTEWSQPKKKRKV 363 transcriptase/SV40 T-antigenPolyarginines R(n); 6 < n < 12 364 MAP KLALKLALKALKAALKLA 365 R6W3RRWWRRWRR 366 NLS CGYGPKKKRKVGG 367 8-lysines KKKKKKKK 368 ARF (1-22)MVRRFLVTLRIRRACGPPRVRV 369 Azurin-p28 LSTAADMQGVVTDGMASGLDKDYLKPDD 370

Linkers

In some embodiments, a linker described herein is a cleavable linker ora non-cleavable linker. In some instances, the linker is a cleavablelinker. In other instances, the linker is a non-cleavable linker.

In some cases, the linker is a non-polymeric linker. A non-polymericlinker refers to a linker that does not contain a repeating unit ofmonomers generated by a polymerization process. Exemplary non-polymericlinkers include, but are not limited to, C₁-C₆ alkyl group (e.g., a C₅,C₄, C₃, C₂, or C₁ alkyl group), homobifunctional cross linkers,heterobifunctional cross linkers, peptide linkers, traceless linkers,self-immolative linkers, maleimide-based linkers, or combinationsthereof. In some cases, the non-polymeric linker comprises a C₁-C₆ alkylgroup (e.g. a C₅, C₄, C₃, C₂, or C₁ alkyl group), a homobifunctionalcross linker, a heterobifunctional cross linker, a peptide linker, atraceless linker, a self-immolative linker, a maleimide-based linker, ora combination thereof. In additional cases, the non-polymeric linkerdoes not comprise more than two of the same type of linkers, e.g., morethan two homobifunctional cross linkers, or more than two peptidelinkers. In further cases, the non-polymeric linker optionally comprisesone or more reactive functional groups.

In some instances, the non-polymeric linker does not encompass a polymerthat is described above. In some instances, the non-polymeric linkerdoes not encompass a polymer encompassed by the polymer moiety C. Insome cases, the non-polymeric linker does not encompass a polyalkyleneoxide (e.g., PEG). In some cases, the non-polymeric linker does notencompass a PEG.

In some instances, the linker comprises a homobifunctional linker.Exemplary homobifunctional linkers include, but are not limited to,Lomant's reagent dithiobis (succinimidylpropionate) DSP,3′3′-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidylsuberate (DSS), bis(sulfosuccinimidvl)suberate (BS), disuccinimidyltartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethyleneglycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG),N,N′-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA),dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS),dimethyl-3,3′-dithiobispropionimidate (DTBP),1,4-di-3′-(2′-pyridyldithio)propionamido)butane (DPDPB),bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), suchas e.g. 1,5-difluoro-2,4-dinitrobenzene or1,3-difluoro-4,6-dinitrobenzene, 4,4′-difluoro-3,3′-dinitrophenylsulfone(DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED),formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipicacid dihydrazide, carbohydrazide, o-toluidine, 3,3′-dimethylbenzidine,benzidine, α,α′-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid.N,N′-ethylene-bis(iodoacetamide), orN,N′-hexamethylene-bis(iodoacetamide).

In some embodiments, the linker comprises a heterobifunctional linker.Exemplary heterobifunctional linker include, but are not limited to,amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble-long-chainN-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT),sulfosuccinimidyl-6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate(sulfo-LC-sMPT),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC),sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs),m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs),N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB),sulfosuccinimidyl(4-iodoacteyl)aminobenzoate (sulfo-sIAB),succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB),sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB).N-(Y-maleimidobutyryloxy)succinimide ester (GMBs),N-(f-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs),succinimidyl 6-((iodoacetyl)amino)hexanoate (slAX), succinimidyl6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (slAC),succinimidyl6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino) hexanoate(sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive andsulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyricacid hydrazide (MPBH),4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M₂C₂H),3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive andphotoreactive cross-linkers such asN-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA).N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA),sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA),sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl-1,3′-dithiopropionate(sAsD), N-hydroxysuccinimidyl-4-azidobenzoate (HsAB),N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB),N-succinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate (sANPAH),sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino)hexanoate(sulfo-sANPAH), N-5-azido-2-nitrobenzovloxvsuccinimide (ANB-NOs),sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3′-dithiopropionate(sAND), N-succinimidyl-4(4-azidophenvl)1,3′-dithiopropionate (sADP),N-sulfosuccinimidyl(4-azidophenyl)-1,3′-dithiopropionate (sulfo-sADP),sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB),sulfosuccinimidyl2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-1,3′-dithiopropionate(sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate(sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP),p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP),sulfhydryl-reactive and photoreactive cross-linkers such as1-(ρ-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB),N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionanmide(APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidecarbonyl-reactive and photoreactive cross-linkers such as p-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive cross-linkerssuch as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactiveand photoreactive cross-linkers such as ρ-azidophenyl glyoxal (APG).

In some instances, the linker comprises a reactive functional group. Insome cases, the reactive functional group comprises a nucleophilic groupthat is reactive to an electrophilic group present on a binding moiety.Exemplary electrophilic groups include carbonyl groups-such as aldehyde,ketone, carboxylic acid, ester, amide, enone, acyl halide or acidanhydride. In some embodiments, the reactive functional group isaldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino,hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazine.

In some embodiments, the linker comprises a maleimide group. In someinstances, the maleimide group is also referred to as a maleimidespacer. In some instances, the maleimide group further encompasses acaproic acid, forming maleimidocaproyl (mc). In some cases, the linkercomprises maleimidocaproyl (mc). In some cases, the linker ismaleimidocaproyl (mc). In other instances, the maleimide group comprisesa maleimidomethyl group, such assuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) orsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(sulfo-sMCC) described above.

In some embodiments, the maleimide group is a self-stabilizingmaleimide. In some instances, the self-stabilizing maleimide utilizesdiaminopropionic acid (DPR) to incorporate a basic amino group adjacentto the maleimide to provide intramolecular catalysis of tiosuccinimidering hydrolysis, thereby eliminating maleimide from undergoing anelimination reaction through a retro-Michael reaction. In someinstances, the self-stabilizing maleimide is a maleimide group describedin Lyon et al., “Self-hydrolyzing maleimides improve the stability andpharmacological properties of antibody-drug conjugates,” Nat.Biotechnol. 32(10):1059-1062 (2014). In some instances, the linkercomprises a self-stabilizing maleimide. In some instances, the linker isa self-stabilizing maleimide.

In some embodiments, the linker comprises a peptide moiety. In someinstances, the peptide moiety comprises at least 2, 3, 4, 5, or 6 moreamino acid residues. In some instances, the peptide moiety comprises atmost 2, 3, 4, 5, 6, 7, or 8 amino acid residues. In some instances, thepeptide moiety comprises about 2, about 3, about 4, about 5, or about 6amino acid residues. In some instances, the peptide moiety is acleavable peptide moiety (e.g., either enzymatically or chemically). Insome instances, the peptide moiety is a non-cleavable peptide moiety. Insome instances, the peptide moiety comprises Val-Cit(valine-citrulline), Gly-Gly-Phe-Gly (SEQ ID NO: 403), Phe-Lys, Val-Lys,Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit,Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu (SEQ ID NO: 404), orGly-Phe-Leu-Gly (SEQ ID NO: 410). In some instances, the linkercomprises a peptide moiety such as: Val-Cit (valine-citrulline),Gly-Gly-Phe-Gly, Phe-Lys. Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys.Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala,Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly (SEQ ID NO: 405). In some cases, thelinker comprises Val-Cit. In some cases, the linker is Val-Cit.

In some embodiments, the linker comprises a benzoic acid group, or itsderivatives thereof. In some instances, the benzoic acid group or itsderivatives thereof comprise paraaminobenzoic acid (PABA). In someinstances, the benzoic acid group or its derivatives thereof comprisegamma-aminobutyric acid (GABA).

In some embodiments, the linker comprises one or more of a maleimidegroup, a peptide moiety, and/or a benzoic acid group, in anycombination. In some embodiments, the linker comprises a combination ofa maleimide group, a peptide moiety, and/or a benzoic acid group. Insome instances, the maleimide group is maleimidocaproyl (mc). In someinstances, the peptide group is val-cit. In some instances, the benzoicacid group is PABA. In some instances, the linker comprises a mc-val-citgroup. In some cases, the linker comprises a val-cit-PABA group. Inadditional cases, the linker comprises a mc-val-cit-PABA group.

In some embodiments, the linker is a self-immolative linker or aself-elimination linker. In some cases, the linker is a self-immolativelinker. In other cases, the linker is a self-elimination linker (e.g., acyclization self-elimination linker). In some instances, the linkercomprises a linker described in U.S. Pat. No. 9,089,614 or PCTPublication NO. WO2015038426.

In some embodiments, the linker is a dendritic type linker. In someinstances, the dendritic type linker comprises a branching,multifunctional linker moiety. In some instances, the dendritic typelinker is used to increase the molar ratio of polynucleotide B to thebinding moiety A. In some instances, the dendritic type linker comprisesPAMAM dendrimers.

In some embodiments, the linker is a traceless linker or a linker inwhich after cleavage does not leave behind a linker moiety (e.g., anatom or a linker group) to a binding moiety A, a polynucleotide B, apolymer C, or an endosomolytic moiety D. Exemplary traceless linkersinclude, but are not limited to, germanium linkers, silicium linkers,sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers,boron linkers, chromium linkers, or phenylhydrazide linker. In somecases, the linker is a traceless aryl-triazene linker as described inHejesen, et al., “A traceless aryl-triazene linker for DNA-directedchemistry.” Org Biomol Chem 11(15): 2493-2497 (2013). In some instances,the linker is a traceless linker described in Blaney, et al., “Tracelesssolid-phase organic synthesis,” Chem. Rev. 102: 2607-2024 (2002). Insome instances, a linker is a traceless linker as described in U.S. Pat.No. 6,821,783.

In some instances, the linker is a linker described in U.S. Pat. Nos.6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. PatentPublication NOs. 2014/0127239; 2013/028919; 2014/286970; 2013/0309256;2015/037360; or 2014/0294851; or PCT Publication NOs. WO2015057699;WO2014080251: WO2014197854; WO2014145090; or WO2014177042.

In some embodiments, X₁ and X₂ are each independently a bond or anon-polymeric linker. In some instances, X₁ and X₂ are eachindependently a bond. In some cases, X₁ and X₂ are each independently anon-polymeric linker.

In some instances, X₁ is a bond or a non-polymeric linker. In someinstances, X₁ is a bond. In some instances, X₁ is a non-polymericlinker. In some instances, the linker is a C₁-C₆ alkyl group. In somecases, X₁ is a C₁-C₆ alkyl group, such as for example, a C₅, C₄, C₃, C₂,or C₁ alkyl group. In some cases, the C₁-C₆ alkyl group is anunsubstituted C₁-C₆ alkyl group. As used in the context of a linker, andin particular in the context of X₁, alkyl means a saturated straight orbranched hydrocarbon radical containing up to six carbon atoms. In someinstances, X₁ includes a homobifunctional linker or a heterobifunctionallinker described supra. In some cases, X₁ includes a heterobifunctionallinker. In some cases, X₁ includes sMCC. In other instances. X₁ includesa heterobifunctional linker optionally conjugated to a C₁-C₆ alkylgroup. In other instances, X₁ includes sMCC optionally conjugated to aC₁-C₆ alkyl group. In additional instances, X₁ does not include ahomobifunctional linker or a heterobifunctional linker described supra.

In some instances, X₂ is a bond or a linker. In some instances, X₂ is abond. In other cases, X₂ is a linker. In additional cases, X₂ is anon-polymeric linker. In some embodiments, X₂ is a C₁-C₆ alkyl group. Insome instances, X₂ is a homobifunctional linker or a heterobifunctionallinker described supra. In some instances, X₂ is a homobifunctionallinker described supra. In some instances, X₂ is a heterobifunctionallinker described supra. In some instances, X₂ comprises a maleimidegroup, such as maleimidocaproyl (mc) or a self-stabilizing maleimidegroup described above. In some instances, X₂ comprises a peptide moiety,such as Val-Cit. In some instances, X₂ comprises a benzoic acid group,such as PABA. In additional instances, X₂ comprises a combination of amaleimide group, a peptide moiety, and/or a benzoic acid group. Inadditional instances, X₂ comprises a mc group. In additional instances,X₂ comprises a mc-val-cit group. In additional instances, X₂ comprises aval-cit-PABA group. In additional instances, X₂ comprises amc-val-cit-PABA group.

Methods of Use

Muscle atrophy refers to a loss of muscle mass and/or to a progressiveweakening and degeneration of muscles. In some cases, the loss of musclemass and/or the progressive weakening and degeneration of muscles occursdue to a high rate of protein degradation, a low rate of proteinsynthesis, or a combination of both. In some cases, a high rate ofmuscle protein degradation is due to muscle protein catabolism (i.e.,the breakdown of muscle protein in order to use amino acids assubstrates for gluconeogenesis).

In one embodiment, muscle atrophy refers to a significant loss in musclestrength. By significant loss in muscle strength is meant a reduction ofstrength in diseased, injured, or unused muscle tissue in a subjectrelative to the same muscle tissue in a control subject. In anembodiment, a significant loss in muscle strength is a reduction instrength of at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, ormore relative to the same muscle tissue in a control subject. In anotherembodiment, by significant loss in muscle strength is meant a reductionof strength in unused muscle tissue relative to the muscle strength ofthe same muscle tissue in the same subject prior to a period of nonuse.In an embodiment, a significant loss in muscle strength is a reductionof at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, or more relativeto the muscle strength of the same muscle tissue in the same subjectprior to a period of nonuse.

In another embodiment, muscle atrophy refers to a significant loss inmuscle mass. By significant loss in muscle mass is meant a reduction ofmuscle volume in diseased, injured, or unused muscle tissue in a subjectrelative to the same muscle tissue in a control subject. In anembodiment, a significant loss of muscle volume is at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, or more relative to the samemuscle tissue in a control subject. In another embodiment, bysignificant loss in muscle mass is meant a reduction of muscle volume inunused muscle tissue relative to the muscle volume of the same muscletissue in the same subject prior to a period of nonuse. In anembodiment, a significant loss in muscle tissue is at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, or more relative to the musclevolume of the same muscle tissue in the same subject prior to a periodof nonuse. Muscle volume is optionally measured by evaluating thecross-section area of a muscle such as by Magnetic Resonance Imaging(e.g. by a muscle volume/cross-section area (CSA) MRI method).

In some embodiments, described herein is a method of treating muscleatrophy in a subject, which comprises providing polynucleic acidmolecule described herein and administering to the subject atherapeutically effective amount of a polynucleic acid moleculedescribed herein or a polynucleic acid molecule conjugate describedherein to reduces a quantity of the mRNA transcript of human DUX4. Insome instances, the muscle atrophy is associated withFacioscapulohumeral muscular dystrophy (FSHD). The polynucleic acidmoiety mediates RNA interference against the human DUX4 as to modulatingmuscle atrophy in a subject. In some embodiments, expression of one ormore marker genes that are affected by DUX4 expression is also alteredor modulated (e.g., decreased) by the decreased expression of humanDUX4. The marker genes includes, but not limited to, MBD3L2, TRIM43,PRAMEF1, ZSCAN4, KHDC1L, and LEUTX. In some embodiments, the expressionof one or more marker genes is decreased at least 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, or 50% compared to untreated cells. In someembodiments, the expression of one or more marker genes, as a group or acomposite, is decreased at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,or 50% compared to untreated cells.

In some embodiments, described herein is a method of treating muscleatrophy in a subject, which comprises providing an siRNA antibodyconjugate described herein and administering to the subject atherapeutically effective amount of the siRNA antibody conjugatedescribed herein and reducing the levels of mRNA transcript of humanDUX4 in said subject. In some instances, the muscle atrophy isassociated with FSHD. The siRNA antibody conjugate mediates RNAinterference against the human DUX4 mRNA as to treat muscle atrophy inthe subject, which comprises administering to the subject atherapeutically effective amount of the siRNA antibody conjugatedescribed herein and reducing the levels of mRNA transcript of humanDUX4 in said subject.

In some embodiments, described herein is a method of treating muscleatrophy in a subject, which comprises providing a DUX4siRNA antibodyconjugate (DUX4 siRNA-conjugate or DUX4-AOC) described herein andadministering to the subject a therapeutically effective amount of theDUX4 siRNA antibody conjugate described herein and reducing the levelsof mRNA transcript of human DUX4 in said subject. In some instances, themuscle atrophy is associated with FSHD. The DUX4 siRNA antibodyconjugate mediates RNA interference against the human DUX4 mRNA as totreat muscle atrophy in the subject, which comprises administering tothe subject a therapeutically effective amount of the DUX4 siRNAantibody conjugate described herein and reducing the levels of mRNAtranscript of human DUX4 in said subject.

In some embodiments, described herein is a method of treating FSHD in asubject, which comprises providing a DUX4 siRNA antibody conjugate (DUX4siRNA conjugate or DUX4-AOC) described herein and administering to thesubject a therapeutically effective amount of the DUX4 siRNA antibodyconjugate described herein and reducing the levels of mRNA transcript ofhuman DUX4 in said subject. In some instances, the FSHD is FSHD type 1(FSHD1). In some instances, the FSHD is FSHD type 2. The DUX4 siRNAantibody conjugate mediates RNA interference against the human DUX4 mRNAas to treat FSHD in the subject, which comprises administering to thesubject a therapeutically effective amount of the DUX4 siRNA-conjugatedescribed herein and reducing the levels of mRNA transcript of humanDUX4 in said subject. In some embodiments, expression levels of one ormore marker genes that are affected by DUX4 expression are also alteredor modulated by the decreased expression levels of human DUX4. The DUX4biomarker genes include but are not limited to MBD3L2, TRIM43, PRAMEF1,ZSCAN4, KHDCIL, and LEUTX.

In some embodiments, described herein is a method of alleviatingsymptoms in a subject with FSHD, which comprises providing a DUX siRNAantibody conjugate (DUX4-siRNA conjugate or DUX4-AOC) described hereinand administering to the subject a therapeutically effective amount ofthe siRNA conjugate described herein by reducing the levels of mRNAtranscript of human DUX4. In some instances, the FSHD is FSHD type 1(FSHD1). In some instances, the FSHD is FSHD type 2. In anotherembodiments, described herein is a method of alleviating symptoms in aFSHD patient, which comprises providing an siRNA conjugate describedherein and administering to the FSHD patient a therapeutically effectiveamount of the siRNA conjugate describes herein by reducing the levels ofmRNA transcript of human DUX4 or reducing the levels of DUX4 protein.

In some instances, the symptoms of FSHD affect skeletal muscles. Theskeletal muscles affected by FSHD include muscles around the eyes andmouth, muscle of the shoulders, muscle of the upper arms, muscle of thelower legs, abdominal muscles and hip muscles. In some instances, thesymptoms of FSHD also affects vision and hearing. In some instances, thesymptoms of FSHD also affect the function of the heart or lungs. In someinstances, the symptoms of FSHD include muscle weakness, muscle atrophy,muscle dystrophy, pain inflammation, contractures, scoliosis, lordosis,hypoventilation, abnormalities of the retina, exposure to keratitis,mild hearing loss, and EMG abnormality.

In some embodiments, described herein is a method of improving skeletalmuscle functions in a FSHD patient comprising the step of administeringto the FSHD patient a therapeutically effective amount of the siRNAconjugate described herein by reducing the levels of mRNA transcript ofhuman DUX4 or reducing the levels of DUX4 protein. In some instances,FSHD is FSHD type 1 (FSHD1). In some instances, FSHD is FSHD type 2. Insome embodiments, described herein is a method of improving skeletalmuscle functions, vision, hearing, heart functions or lung functions ina patient suffering from FSHD comprising the step of administering tothe FSHD patient a therapeutically effective amount of the siRNAconjugate described herein by reducing the levels of mRNA transcript ofhuman DUX4 or reducing the levels of DUX4 protein.

In some embodiments, described herein is a method of treating FSHD in asubject, which comprises providing an antisense oligonucleotide (ASO)antibody conjugate (ASO conjugate) described herein and administering tothe subject a therapeutically effective amount of the ASO-antibodyconjugate described herein and reducing the levels of mRNA transcript ofhuman DUX4 in said subject. In some instances, FSHD is FSHD type 1(FSHD1). In some instances, FSHD is FSHD type 2. The ASO-conjugatemediates RNA interference against the human DUX4 mRNA as to treat FSHDin the subject, which comprises administering to the subject atherapeutically effective amount of the ASO-antibody conjugate describedherein and reducing the levels of mRNA transcript of human DUX4 in saidsubject. In some embodiments, expression levels of one or more markergenes that are affected by DUX4 expression is also altered or modulatedby the decreased expression levels of human DUX4. The DUX4 biomarkergenes include but are not limited to MBD3L2, TRIM43, PRAMEF1, ZSCAN4,KHDC1L, and LEUTX.

In some embodiments, described herein is a method of treating FSHD in asubject. In some instances, the FSHD subject suffers from FSHD1. Inother instances, the FSHD subject suffers from FSHD2. In anotherembodiment, the FSHD subject has muscle cells abnormally expressing DUX4protein caused by the genetic and epigenetic molecular changes in theD4Z4 region of the long arm of chromosome 4. The genetic molecularchanges in the muscle cells are mutations leading to the contraction ofthe D4Z4 region containing 1-10 repeats instead of the normal 11 to 100repeats of chromosome 4 of the FSHD subject. The epigenetic molecularchanges in the muscle cells are changes leading to the hypomethylationof the D4Z4 region of chromosome 4 of the FSHD subject. In someinstances, the muscle cells are skeletal muscle cells.

Pharmaceutical Formulation

In some embodiments, the pharmaceutical formulations described hereinare administered to a subject by multiple administration routes,including but not limited to, parenteral (e.g., intravenous,subcutaneous, intramuscular), oral, intranasal, buccal, rectal, ortransdermal administration routes. In some instances, the pharmaceuticalcomposition describe herein is formulated for parenteral (e.g.,intravenous, subcutaneous, intramuscular, intra-arterial,intraperitoneal, intrathecal, intracerebral, intracerebroventricular, orintracranial) administration. In other instances, the pharmaceuticalcomposition describe herein is formulated for oral administration. Instill other instances, the pharmaceutical composition describe herein isformulated for intranasal administration.

In some embodiments, the pharmaceutical formulations include, but arenot limited to, aqueous liquid dispersions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate release formulations, controlledrelease formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations (e.g., nanoparticleformulations), and mixed immediate and controlled release formulations.

In some instances, the pharmaceutical formulation includesmultiparticulate formulations. In some instances, the pharmaceuticalformulation includes nanoparticle formulations. In some instances,nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases,nanoparticles comprise solid lipid nanoparticles, polymericnanoparticles, self-emulsifying nanoparticles, liposomes,microemulsions, or micellar solutions. Additional exemplarynanoparticles include, but are not limited to, paramagneticnanoparticles, superparamagnetic nanoparticles, metal nanoparticles,fullerene-like materials, inorganic nanotubes, dendrimers (such as withcovalently attached metal chelates), nanofibers, nanohoms, nano-onions,nanorods, nanoropes and quantum dots. In some instances, a nanoparticleis a metal nanoparticle, e.g., a nanoparticle of scandium, titanium,vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc,yttrium, zirconium, niobium, molybdenum, ruthenium, rhodium, palladium,silver, cadmium, hafnium, tantalum, tungsten, rhenium, osmium, iridium,platinum, gold, gadolinium, aluminum, gallium, indium, tin, thallium,lead, bismuth, magnesium, calcium, strontium, barium, lithium, sodium,potassium, boron, silicon, phosphorus, germanium, arsenic, antimony, andcombinations, alloys or oxides thereof.

In some instances, a nanoparticle includes a core or a core and a shell,as in a core-shell nanoparticle.

In some instances, a nanoparticle is further coated with molecules forattachment of functional elements (e.g., with one or more of apolynucleic acid molecule or binding moiety described herein). In someinstances, a coating comprises chondroitin sulfate, dextran sulfate,carboxymethyl dextran, alginic acid, pectin, carragheenan, fucoidan,agaropectin, porphyran, karaya gum, gellan gum, xanthan gum, hyaluronicacids, glucosamine, galactosamine, chitin (or chitosan), polyglutamicacid, polyaspartic acid, lysozyme, cytochrome C, ribonuclease,trypsinogen, chymotrypsinogen, α-chymotrypsin, polylysine, polyarginine,histone, protamine, ovalbumin or dextrin or cyclodextrin. In someinstances, a nanoparticle comprises a graphene-coated nanoparticle.

In some cases, a nanoparticle has at least one dimension of less thanabout 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm.

In some instances, the nanoparticle formulation comprises paramagneticnanoparticles, superparamagnetic nanoparticles, metal nanoparticles,fullerene-like materials, inorganic nanotubes, dendrimers (such as withcovalently attached metal chelates), nanofibers, nanohorns, nano-onions,nanorods, nanoropes or quantum dots. In some instances, a polynucleicacid molecule or a binding moiety described herein is conjugated eitherdirectly or indirectly to the nanoparticle. In some instances, at least1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more polynucleicacid molecules or binding moieties described herein are conjugatedeither directly or indirectly to a nanoparticle.

In some embodiments, the pharmaceutical formulation comprises a deliveryvector, e.g., a recombinant vector, the delivery of the polynucleic acidmolecule into cells. In some instances, the recombinant vector is DNAplasmid. In other instances, the recombinant vector is a viral vector.Exemplary viral vectors include vectors derived from adeno-associatedvirus, retrovirus, adenovirus, or alphavirus. In some instances, therecombinant vectors capable of expressing the polynucleic acid moleculesprovide stable expression in target cells. In additional instances,viral vectors are used that provide for transient expression ofpolynucleic acid molecules.

In some embodiments, the pharmaceutical formulation includes a carrieror carrier materials selected on the basis of compatibility with thecomposition disclosed herein, and the release profile properties of thedesired dosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. Pharmaceutically compatible carrier materials include, but are notlimited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical DosageForms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical DosageForms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams &Wilkins 1999).

In some instances, the pharmaceutical formulation further includes pHadjusting agents or buffering agents which include acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In some instances, the pharmaceutical formulation includes one or moresalts in an amount required to bring osmolality of the composition intoan acceptable range. Such salts include those having sodium, potassiumor ammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

In some instances, the pharmaceutical formulation further includesdiluent which are used to stabilize compounds because they provide amore stable environment. Salts dissolved in buffered solutions (whichalso provide pH control or maintenance) are utilized as diluents in theart, including, but not limited to a phosphate buffered saline solution.In certain instances, diluents increase bulk of the composition tofacilitate compression or create sufficient bulk for homogenous blendfor capsule filling. Such compounds include e.g., lactose, starch,mannitol, sorbitol, dextrose, microcrystalline cellulose such asAvicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate;tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-driedlactose; pregelatinized starch, compressible sugar, such as Di-Pac®(Amstar); mannitol, hydroxypropylmethylcellulose,hydroxypropylmethylcellulose acetate stearate, sucrose-based diluents,confectioner's sugar; monobasic calcium sulfate monohydrate, calciumsulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzedcereal solids, amylose; powdered cellulose, calcium carbonate; glycine,kaolin; mannitol, sodium chloride; inositol, bentonite, and the like.

In some cases, the pharmaceutical formulation includes disintegrationagents or disintegrants to facilitate the breakup or disintegration of asubstance. The term “disintegrate” include both the dissolution anddispersion of the dosage form when contacted with gastrointestinalfluid. Examples of disintegration agents include a starch, e.g., anatural starch such as corn starch or potato starch, a pregelatinizedstarch such as National 1551 or Amijel®, or sodium starch glycolate suchas Promogel® or Explotab®, a cellulose such as a wood product,methylcrvstalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose, a cross-linked starch such as sodium starch glycolate, across-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginicacid such as sodium alginate, a clay such as Veegum® HV (magnesiumaluminum silicate), a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth, sodium starch glycolate, bentonite, a naturalsponge, a surfactant, a resin such as a cation-exchange resin, citruspulp, sodium lauryl sulfate, sodium lauryl sulfate in combinationstarch, and the like.

In some instances, the pharmaceutical formulation includes fillingagents such as lactose, calcium carbonate, calcium phosphate, dibasiccalcium phosphate, calcium sulfate, microcrystalline cellulose,cellulose powder, dextrose, dextrates, dextran, starches, pregelatinizedstarch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,polyethylene glycol, and the like.

Lubricants and glidants are also optionally included in thepharmaceutical formulations described herein for preventing, reducing orinhibiting adhesion or friction of materials. Exemplary lubricantsinclude, e.g., stearic acid, calcium hydroxide, talc, sodium stearylfumerate, a hydrocarbon such as mineral oil, or hydrogenated vegetableoil such as hydrogenated soybean oil (Sterotex®), higher fatty acids andtheir alkali-metal and alkaline earth metal salts, such as aluminum,calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol,talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate,sodium chloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™. Cab-O-Sil®, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

Plasticizers include compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300. PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. Plasticizers alsofunction as dispersing agents or wetting agents.

Solubilizers include compounds such as triacetin, triethylcitrate, ethyloleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

Stabilizers include compounds such as any antioxidation agents, buffers,acids, preservatives and the like.

Suspending agents include compounds such as polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, vinyl pyrrolidone/vinyl acetatecopolymer (S630), polyethylene glycol, e.g., the polyethylene glycol hasa molecular weight of about 300 to about 6000, or about 3350 to about4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose, hydroxymethylcelluloseacetate stearate, polysorbate-80, hydroxyethylcellulose, sodiumalginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum,xanthans, including xanthan gum, sugars, cellulosics, such as, e.g.,sodium carboxymethylcellulose, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose,hydroxyethylcellulose, polysorbate-80, sodium alginate, polyethoxylatedsorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone andthe like.

Surfactants include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g. Pluronic® (BASF), and the like.Additional surfactants include polvoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. Sometimes, surfactants is included to enhance physicalstability or for other purposes.

Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, hydroxypropylmethyl cellulose acetate stearate,hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol,alginates, acacia, chitosans and combinations thereof.

Wetting agents include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

Therapeutic Regimens

In some embodiments, the pharmaceutical compositions described hereinare administered for therapeutic applications. In some embodiments, thepharmaceutical composition is administered once per day, twice per day,three times per day or more. The pharmaceutical composition isadministered daily, every day, every alternate day, five days a week,once a week, every other week, two weeks per month, three weeks permonth, once a month, twice a month, three times per month, once in twomonths, once in three months, once in four months, once in five months,once in six months or more. The pharmaceutical composition isadministered for at least 1 month, 2 months, 3 months, 4 months, 5months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12months, 18 months, 2 years, 3 years, or more.

In some embodiments, one or more pharmaceutical compositions areadministered simultaneously, sequentially, or at an interval period oftime. In some embodiments, one or more pharmaceutical compositions areadministered simultaneously. In some cases, one or more pharmaceuticalcompositions are administered sequentially. In additional cases, one ormore pharmaceutical compositions are administered at an interval periodof time (e.g., the first administration of a first pharmaceuticalcomposition is on day one followed by an interval of at least 1, 2, 3,4, 5, or more days prior to the administration of at least a secondpharmaceutical composition).

In some embodiments, two or more different pharmaceutical compositionsare co-administered. In some instances, the two or more differentpharmaceutical compositions are co-administered simultaneously. In somecases, the two or more different pharmaceutical compositions areco-administered sequentially without a gap of time betweenadministrations. In other cases, the two or more differentpharmaceutical compositions are co-administered sequentially with a gapof about 0.5 hour, 1 hour, 2 hour, 3 hour, 12 hours, 1 day, 2 days, ormore between administrations.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the composition is given continuously;alternatively, the dose of the composition being administered istemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). In some instances, the length of the drugholiday varies between 2 days and 1 year, including by way of exampleonly, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days,15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320days, 350 days, or 365 days. The dose reduction during a drug holiday isfrom 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, can be reduced, as a function ofthe symptoms, to a level at which the improved disease, disorder orcondition is retained.

In some embodiments, the amount of a given agent that correspond to suchan amount varies depending upon factors such as the particular compound,the severity of the disease, the identity (e.g., weight) of the subjector host in need of treatment, but nevertheless is routinely determinedin a manner known in the art according to the particular circumstancessurrounding the case, including, e.g., the specific agent beingadministered, the route of administration, and the subject or host beingtreated. In some instances, the desired dose is conveniently presentedin a single dose or as divided doses administered simultaneously (orover a short period of time) or at appropriate intervals, for example astwo, three, four or more sub-doses per day.

The foregoing ranges are merely suggestive, as the number of variablesin regard to an individual treatment regime is large, and considerableexcursions from these recommended values are not uncommon. Such dosagesis altered depending on a number of variables, not limited to theactivity of the compound used, the disease or condition to be treated,the mode of administration, the requirements of the individual subject,the severity of the disease or condition being treated, and the judgmentof the practitioner.

In some embodiments, toxicity and therapeutic efficacy of suchtherapeutic regimens are determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, including, but notlimited to, the determination of the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between the toxic and therapeuticeffects is the therapeutic index and it is expressed as the ratiobetween LD50 and ED50. Compounds exhibiting high therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesare used in formulating a range of dosage for use in human. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with minimal toxicity. The dosagevaries within this range depending upon the dosage form employed and theroute of administration utilized.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more of the compositions and methodsdescribed herein. Such kits include a carner, package, or container thatis compartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) comprising one of theseparate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. In one embodiment, the containers are formed from a variety ofmaterials such as glass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

For example, the container(s) include target nucleic acid moleculedescribed herein. Such kits optionally include an identifyingdescription or label or instructions relating to its use in the methodsdescribed herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In one embodiment, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

In certain embodiments, the pharmaceutical compositions are presented ina pack or dispenser device which contains one or more unit dosage formscontaining a compound provided herein. The pack, for example, containsmetal or plastic foil, such as a blister pack. In one embodiment, thepack or dispenser device is accompanied by instructions foradministration. In one embodiment, the pack or dispenser is alsoaccompanied with a notice associated with the container in formprescribed by a governmental agency regulating the manufacture, use, orsale of pharmaceuticals, which notice is reflective of approval by theagency of the form of the drug for human or veterinary administration.Such notice, for example, is the labeling approved by the U.S. Food andDrug Administration for prescription drugs, or the approved productinsert. In one embodiment, compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier are alsoprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. It is to be understoodthat the foregoing general description and the following detaileddescription are exemplary and explanatory only and are not restrictiveof any subject matter claimed. In this application, the use of thesingular includes the plural unless specifically stated otherwise. Itmust be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. In this application, theuse of “or” means “and/or” unless stated otherwise. Furthermore, use ofthe term “including” as well as other forms, such as “include”,“includes,” and “included,” is not limiting.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term“about” includes an amount that would be expected to be withinexperimental error.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

As used herein, the terms “individual(s)”, “subject(s)“and” patient(s)”mean any mammal. In some embodiments, the mammal is a human. In someembodiments, the mammal is a non-human. None of the terms require or arelimited to situations characterized by the supervision (e.g. constant orintermittent) of a health care worker (e.g. a doctor, a registerednurse, a nurse practitioner, a physician's assistant, an orderly or ahospice worker).

The term “therapeutically effective amount” relates to an amount of apolynucleic acid molecule conjugate that is sufficient to provide adesired therapeutic effect in a mammalian subject. In some cases, theamount is single or multiple dose administration to a patient (such as ahuman) for treating, preventing, preventing the onset of, curing,delaying, reducing the severity of, ameliorating at least one symptom ofa disorder or recurring disorder, or prolonging the survival of thepatient beyond that expected in the absence of such treatment.Naturally, dosage levels of the particular polynucleic acid moleculeconjugate employed to provide a therapeutically effective amount vary independence of the type of injury, the age, the weight, the gender, themedical condition of the subject, the severity of the condition, theroute of administration, and the particular inhibitor employed. In someinstances, therapeutically effective amounts of polynucleic acidmolecule conjugate, as described herein, is estimated initially fromcell culture and animal models. For example, IC₅₀ values determined incell culture methods optionally serve as a starting point in animalmodels, while ICs values determined in animal models are optionally usedto find a therapeutically effective dose in humans.

Skeletal muscle, or voluntary muscle, is generally anchored by tendonsto bone and is generally used to effect skeletal movement such aslocomotion or in maintaining posture. Although some control of skeletalmuscle is generally maintained as an unconscious reflex (e.g., posturalmuscles or the diaphragm), skeletal muscles react to conscious control.Smooth muscle, or involuntary muscle, is found within the walls oforgans and structures such as the esophagus, stomach, intestines,uterus, urethra, and blood vessels.

Skeletal muscle is further divided into two broad types: Type I (or“slow twitch”) and Type II (or “fast twitch”). Type I muscle fibers aredense with capillaries and are rich in mitochondria and myoglobin, whichgives Type I muscle tissue a characteristic red color. In some cases,Type I muscle fibers carries more oxygen and sustain aerobic activityusing fats or carbohydrates for fuel. Type I muscle fibers contract forlong periods of time but with little force. Type II muscle fibers arefurther subdivided into three major subtypes (IIa, lIx, and IIb) thatvary in both contractile speed and force generated. Type II musclefibers contract quickly and powerfully but fatigue very rapidly, andtherefore produce only short, anaerobic bursts of activity before musclecontraction becomes painful.

Unlike skeletal muscle, smooth muscle is not under conscious control.

Cardiac muscle is also an involuntary muscle but more closely resemblesskeletal muscle in structure and is found only in the heart. Cardiac andskeletal muscles are striated in that they contain sarcomeres that arepacked into highly regular arrangements of bundles. By contrast, themyofibrils of smooth muscle cells are not arranged in sarcomeres andtherefore are not striated.

Muscle cells encompass any cells that contribute to muscle tissue.Exemplary muscle cells include myoblasts, satellite cells, myotubes, andmyofibril tissues.

As used here, muscle force is proportional to the cross-sectional area(CSA), and muscle velocity is proportional to muscle fiber length. Thus,comparing the cross-sectional areas and muscle fibers between variouskinds of muscles is capable of providing an indication of muscleatrophy. Various methods are known in the art to measure muscle strengthand muscle weight, see, for example, “Musculoskeletal assessment; Jointrange of motion and manual muscle strength” by Hazel M. Clarkson,published by Lippincott Williams & Wilkins, 200). The production oftomographic images from selected muscle tissues by computed axialtomography and sonographic evaluation are additional methods ofmeasuring muscle mass.

The term antibody oligonucleotide conjugate (AOC) refers to an antibodyconjugated to a nucleotide.

The term siRNA conjugate or siRNA antibody conjugate refers to anantibody conjugated to a siRNA.

The term DUX4 siRNA-conjugate or DUX4 siRNA antibody conjugate refers toan antibody conjugated to a siRNA hybridizing to a target sequence ofthe human DUX4 mRNA.

The term DUX4-AOC refers to an antibody conjugated to an siRNAhybridizing to a target sequence of the human DUX4 mRNA.

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1. Bioinformatic siRNA Library Design Against Human Full LengthDUX4 Transcript

FIG. 2 shows a flowchart of in silico selection process of DUX4 siRNA.Sequences of all siRNAs that can binds to DUX4, or a pre-determinedregion of the DUX4 are collected to generate a starting set of DUX4siRNA. From the starting set of DUX siRNAs, the first eliminating stepcomprises eliminating one or more DUX siRNAs that has single nucleotidepolymorphism (SNP) and/or MEF<−5. Then, the second eliminating stepcomprises eliminating DUX siRNAs with 0 and 1 MM in the humantranscriptome (such that only hits allowed are DUX, DUX5, and DBET).Then, the third eliminating step comprises eliminating DUX siRNAs with 0mismatch (MM) in the human intragenic regions (such that only hitsallowed are DUX1, DUX5 and DBET pseudogenes). Then, the next eliminatingstep comprises eliminating DUX siRNAs with a MM to DUX4 human sequenceused in FLExDUX4 FSHD mouse model. Then, the next step is carryingforward only or one or more DUX siRNAs with predicted viability>60.Next, the eliminating step comprises eliminating one or more DUX siRNAswith a match to a seed region of known miRNAs 1-1000. Then, theeliminating step continues with eliminating DUX siRNAs molecule with %GC content 75 and above. Then, the final selection process compriseswith 8 or less predicted off-target hits with 2 MM, except for theregion 295-1132, for which up to 12 hits are allowed. Using such seriesof selection steps, final 70 candidate DUX siRNAs could be selected froma starting set of 1694 DUX siRNAs. FIG. 3 shows the location and numbersof such selected DUX4 siRNA in the DUX4 mRNA transcript (NM_001306068).

Identified siRNA candidates share common characteristics in theirsequences as shown below Table 10 The identified siRNAs have mostly2′-O-Me modifications, with 2′-F modifications only located on sensestrand at positions 7, 8, 9 and 2-′F only located on antisense strand atpositions 1, 2, 6, 14, 16. Also, the identified siRNAs comprises 4thioate modifications on each strand, located at the final 2 linkages ofeach 5′ and 3′ terminus. The identified siRNAs further comprises “Uf” atthe first position of 5′ end of the antisense strand, regardless of theactual target mRNA sequence (coupled with “a” at the last position atthe 3′ end of the sense strand). The identified siRNAs further comprises“uu” overhang at the 3′ end of the antisense strand only, with nooverhang at the 3′ end of the sense strand. The optimization of theidentified siRNAs may comprise a vinyl phosphonate nucleotide, aninverted abasic moiety, or an amine linker to the passenger strand orthe guide strand.

TABLE 10 sense strand antisense strand duplex sequence (5′-3′)sequence (5′-3′) name (passenger strand) (guide strand) DUX4nsnsnnnnNfNfNfnnnnn UfsNfsnnnNfnnnnnnn template nnnsnsa NfnNfnnnsusu vpN= vinyl phosphonate 2′-MOE; upper case (N) = 2′-OH (ribo); lower case(n) = 2′-O-Me (methyl) dN = 2′-H (deoxy); Nf = 2′-F (fluoro); s= phosphorothioate backbone modification; iB = inverted abasic

Tables 11, 12, and 13 illustrate identified siRNA candidates for theregulation of human DUX4.

TABLE 11 19mer start SEQ ID sense/passenger_seq SEQ IDantisense/guide_seq Name site NO (5′-3′) NO (5′-3′) NM_001306068_11_2911 1 cgacaccctcggacagcac 71 gtgctgtccgagggtgtcg NM_001306068_57_75 57 2acggcgacggagactcgtt 72 aacgagtctccgtcgccgt NM_001306068_58_76 58 3cggcgacggagactcgttt 73 aaacgagtctccgtcgccg NM_001306068_59_77 59 4ggcgacggagactcgtttg 74 caaacgagtctccgtcgcc NM_001306068_60_78 60 5gcgacggagactcgtttgg 75 ccaaacgagtctccgtcgc NM_001306068_61_79 61 6cgacggagactcgtttgga 76 tccaaacgagtctccgtcg NM_001306068_62_80 62 7gacggagactcgtttggac 77 gtccaaacgagtctccgtc NM_001306068_63_81 63 8acggagactcgtaggacc 78 ggtccaaacgagtctccgt NM_001306068_77_95 77 9ggaccccgagccaaagcga 79 tcgctttggctcggggtcc NM_001306068_78_96 78 10gaccccgagccaaagcgag 80 ctcgctttggctcggggtc NM_001306068_79_97 79 11accccgagccaaagcgagg 81 cctcgctttggctcggggt NM_001306068_99_117 99 12cctgcgagcctgctttgag 82 ctcaaagcaggctcgcagg NM_001306068_102_120 102 13gcgagcctgctttgagcgg 83 ccgctcaaagcaggctcgc NM_001306068_137_155 137 14tcgccaccagagaacggct 84 agccgttctctggtggcga NM_001306068_160_178 160 15caggccatcggcattccgg 85 ccggaatgccgatggcctg NM_001306068_162_180 162 16ggccatcggcattccggag 86 ctccggaatgccgatggcc NM_001306068_163_181 163 17gccatcggcattccggagc 87 gctccggaatgccgatggc NM_001306068_231_249 231 18gcaccggcgggaatctcgg 88 ccgagattcccgccggtgc NM_001306068_232_250 232 19caccggcgggaatctcggc 89 gccgagattcccgccggtg NM_001306068_274_292 274 20ccagaaggccggcgaaagc 90 gctttcgccggccttctgg NM_001306068_276_294 276 21agaaggccggcgaaagcgg 91 ccgctttcgccggccttct NM_001306068_277_295 277 22gaaggccggcgaaagcgga 92 tccgctttcgccggccttc NM_001306068_285_303 285 23gcgaaagcggaccgccgtc 93 gacggcggtccgctttcgc NM_001306068_287_305 287 24gaaagcggaccgccgtcac 94 gtgacggcggtccgctttc NM_001306068_292_310 292 25cggaccgccgtcaccggat 95 atccggtgacggcggtccg NM_001306068_293_311 293 26ggaccgccgtcaccggatc 96 gatccggtgacggcggtcc NM_001306068_294_312 294 27gaccgccgtcaccggatcc 97 ggatccggtgacggcggtc NM_001306068_389_407 389 28agacgggcctcccggagtc 98 gactccgggaggcccgtct NM_001306068_524_542 524 29cctcgtgggtcgccttcgc 99 gcgaaggcgacccacgagg NM_001306068_525_543 525 30ctcgtgggtcgccttcgcc 100 ggcgaaggcgacccacgag NM_001306068_679_697 679 31gaggggatctcccaacctg 101 caggttgggagatcccctc NM_001306068_704_722 704 32cgcgcggggatttcgccta 102 taggcgaaatccccgcgcg NM_001306068_705_723 705 33gcgcggggatttcgcctac 103 gtaggcgaaatccccgcgc NM_001306068_708_726 708 34cggggatttcgcctacgcc 104 ggcgtaggcgaaatccccg NM_001306068_893_911 893 35tgcttgcgccacccacgtc 105 gacgtgggtggcgcaagca NM_001306068_1132_1150 113236 ctggcgagcccggagtttc 106 gaaactccgggctcgccag NM_001306068_1134_11521134 37 ggcgagcccggagtttctg 107 cagaaactccgggctcgccNM_001306068_1158_1176 1158 38 ggcgcaacctctcctagaa 108ttctaggagaggttgcgcc NM_001306068_1159_1177 1159 39 gcgcaacctctcctagaaa109 tttctaggagaggttgcgc NM_001306068_1163_1181 1163 40aacctctcctagaaacgga 110 tccgtttctaggagaggtt NM_001306068_1236_1254 123641 cagcgaggaagaataccgg 111 ccggtattcttcctcgctg NM_001306068_1237_12551237 42 agcgaggaagaataccggg 112 cccggtattcttcctcgctNM_001306068_1238_1256 1238 43 gcgaggaagaataccgggc 113gcccggtattcttcctcgc NM_001306068_1284_1302 1284 44 gttgggacggggtcgggtg114 cacccgaccccgtcccaac NM_001306068_1290_1308 1290 45acggggtcgggtggttcgg 115 ccgaaccacccgaccccgt NM_001306068_1294_1312 129446 ggtcgggtggttcggggca 116 tgccccgaaccacccgacc NM_001306068_1295_13131295 47 gtcgggtggttcggggcag 117 ctgccccgaaccacccgacNM_001306068_1315_1333 1315 48 gcggtggcctctctttcgc 118gcgaaagagaggccaccgc NM_001306068_1316_1334 1316 49 cggtggcctctctttcgcg119 cgcgaaagagaggccaccg NM_001306068_1317_1335 1317 50ggtggcctctctttcgcgg 120 ccgcgaaagagaggccacc NM_001306068_1321_1339 132151 gcctctctttcgcggggaa 121 ttccccgcgaaagagaggc NM_001306068_1340_13581340 52 cacctggctggctacggag 122 ctccgtagccagccaggtgNM_001306068_1350_1368 1350 53 gctacggaggggcgtgtct 123agacacgcccctccgtagc NM_001306068_1351_1369 1351 54 ctacggaggggcgtgtctc124 gagacacgcccctccgtag NM_001306068_1539_1557 1539 55acgtgcaagggagctcgct 125 agcgagctcccttgcacgt NM_001306068_1540_1558 154056 cgtgcaagggagctcgctg 126 cagcgagctcccttgcacg NM_001306068_1541_15591541 57 gtgcaagggagctcgctgg 127 ccagcgagctcccttgcacNM_001306068_1610_1628 1610 58 caccttccgacgctgtcta 128tagacagcgtcggaaggtg NM_001306068_1611_1629 1611 59 accttccgacgctgtctag129 ctagacagcgtcggaaggt NM_001306068_1612_1630 1612 60ccttccgacgctgtctagg 130 cctagacagcgtcggaagg NM_001306068_1613_1631 161361 cttccgacgctgtctaggc 131 gcctagacagcgtcggaag NM_001306068_1615_16331615 62 tccgacgctgtctaggcaa 132 ttgcctagacagcgtcggaNM_001306068_1616_1634 1616 63 ccgacgctgtctaggcaaa 133tttgcctagacagcgtcgg NM_001306068_1619_1637 1619 64 acgctgtctaggcaaacct134 aggtttgcctagacagcgt NM_001306068_1632_1650 1632 65aaacctggattagagttac 135 gtaactctaatccaggttt NM_001306068_336_354 336 66ctttgagaaggatcgcttt 136 aaagcgatccttctcaaag NM_001306068_672_690 672 67gccggcagaggggatctcc 137 ggagatcccctctgccggc NM_001306068_882_900 882 68gggccaaggggtgcttgcg 138 cgcaagcaccccttggccc NM_001306068_884_902 884 69gccaaggggtgcttgcgcc 139 ggcgcaagcaccccttggc NM_001306068_1045_1063 104570 atgcaaggcatcccggcgc 140 gcgccgggatgccttgcat

TABLE 12 19mer start SEQ ID sense/passenger_seq SEQ IDantisense/guide_seq Name site NO (5′-3′) NO (5′-3′) NM_001306068_11_2911 141 csgsacacCfCfUfcggac 211 UfsUfsgcuGfuccgagg agcsasa GfuGfucgsusuNM_001306068_57_75 57 142 ascsggcgAfCfGfgagac 212 UfsAfscgaGfucuccguucgsusa CfgCfcgususu NM_001306068_58_76 58 143 csgsgcgaCfGfGfagacu 213UfsAfsacgAfgucuccg cgususa UfcGfccgsusu NM_001306068_59_77 59 144gsgscgacGfGfAfgacuc 214 UfsAfsaacGfagucucc guususa GfuCfgccsusuNM_001306068_60_78 60 145 gscsgacgGfAfGfacucg 215 UfsCfsaaaCfgagucucCuuusgsa fgUfcgcsusu NM_001306068_61_79 61 146 csgsacggAfGfAfcucgu 216UfsCfscaaAfcgagucuC uugsgsa fcGfucgsusu NM_001306068_62_80 62 147gsascggaGfAfCfucguu 217 UfsUfsccaAfacgaguc uggsasa UfcCfgucsusuNM_001306068_63_81 63 148 ascsggagAfCfUfcguuu 218 UfsGfsuccAfaacgaguggascsa CfuCfcgususu NM_001306068_77_95 77 149 gsgsacccCfGfAfgccaa 219UfsCfsgcuUfuggcucg agcsgsa GfgGfuccsusu NM_001306068_78_96 78 150gsasccccGfAfGfccaaa 220 UfsUfscgcUfuuggcuc gcgsasa GfgGfgucsusuNM_001306068_79_97 79 151 ascscccgAfGfCfcaaag 221 UfsCfsucgCfuuuggcucgasgsa CfgGfggususu NM_001306068_99_117 99 152 cscsugcgAfGfCfcugcu 222UfsUfscaaAfgcaggcu uugsasa CfgCfaggsusu NM_001306068_102_120 102 153gscsgagcCfUfGfcuuug 223 UfsCfsgcuCfaaagcagG agcsgsa fcUfcgcsusuNM_001306068_137_155 137 154 uscsgccaCfCfAfgagaa 224 UfsGfsccgUfucucuggcggscsa UfgGfcgasusu NM_001306068_160_178 160 155 csasggccAfUfCfggcau225 UfsCfsggaAfugccgau uccsgsa GfgCfcugsusu NM_001306068_162_180 162 156gsgsccauCfGfGfcauuc 226 UfsUfsccgGfaaugccg cggsasa AfuGfgccsusuNM_001306068_163_181 163 157 gscscaucGfGfCfauucc 227 UfsCfsuccGfgaaugccggasgsa GfaUfggcsusu NM_001306068_231_249 231 158 gscsaccgGfCfGfggaau228 UfsCfsgagAfuucccgc cucsgsa CfgGfugcsusu NM_001306068_232_250 232 159csasccggCfGfGfgaauc 229 UfsCfscgaGfauucccgC ucgsgsa fcGfgugsusuNM_001306068_274_292 274 160 cscsagaaGfGfCfcggcg 230 UfsCfsuuuCfgccggccaaasgsa UfuCfuggsusu NM_001306068_276_294 276 161 asgsaaggCfCfGfgcgaa231 UfsCfsgcuUfucgccgg agcsgsa CfcUfucususu NM_001306068_277_295 277 162gsasaggcCfGfGfcgaaa 232 UfsCfscgcUfuucgccg gcgsgsa GfcCfuucsusuNM_001306068_285_303 285 163 gscsgaaaGfCfGfgaccg 233 UfsAfscggCfgguccgcccgsusa UfuUfcgcsusu NM_001306068_287_305 287 164 gsasaagcGfGfAfccgcc234 UfsUfsgacGfgcggucc gucsasa GfcUfuucsusu NM_001306068_292_310 292 165csgsgaccGfCfCfgucac 235 UfsUfsccgGfugacggc cggsasa GfgUfccgsusuNM_001306068_293_311 293 166 gsgsaccgCfCfGfucacc 236 UfsAfsuccGfgugacggggasusa CfgGfuccsusu NM_001306068_294_312 294 167 gsasccgcCfGfUfcaccg237 UfsGfsaucCfggugacg gauscsa GfcGfgucsusu NM_001306068_389_407 389 168asgsacggGfCfCfucccg 238 UfsAfscucCfgggaggc gagsusa CfcGfucususuNM_001306068_524_542 524 169 cscsucguGfGfGfucgcc 239 UfsCfsgaaGfgcgacccAuucsgsa fcGfaggsusu NM_001306068_525_543 525 170 csuscgugGfGfUfcgccu 240UfsGfscgaAfggcgacc ucgscsa CfaCfgagsusu NM_001306068_679_697 679 171gsasggggAfUfCfuccca 241 UfsAfsgguUfgggagau accsusa CfcCfcucsusuNM_001306068_704_722 704 172 csgscgcgGfGfGfauuuc 242 UfsAfsggcGfaaaucccCgccsusa fgCfgcgsusu NM_001306068_705_723 705 173 gscsgcggGfGfAfuuuc 243UfsUfsaggCfgaaauccC gccusasa fcGfcgcsusu NM_001306068_708_726 708 174csgsgggaUfUfUfcgccu 244 UfsGfscguAfggcgaaa acgscsa UfcCfccgsusuNM_001306068_893_911 893 175 usgscuugCfGfCfcaccc 245 UfsAfscguGfgguggcgacgsusa CfaAfgcasusu NM_001306068_1132_1150 1132 176 csusggcgAfGfCfccgga246 UfsAfsaacUfccgggcu guususa CfgCfcagsusu NM_001306068_1134_1152 1134177 gsgscgagCfCfCfggagu 247 UfsAfsgaaAfcuccggg uucsusa CfuCfgccsusuNM_001306068_1158_1176 1158 178 gsgscgcaAfCfCfucucc 248UfsUfscuaGfgagaggu uagsasa UfgCfgccsusu NM_001306068_1159_1177 1159 179gscsgcaaCfCfUfcuccu 249 UfsUfsucuAfggagagg agasasa UfuGfcgcsusuNM_001306068_1163_1181 1163 180 asasccucUfCfCfuagaa 250UfsCfscguUfucuagga acgsgsa GfaGfguususu NM_001306068_1236_1254 1236 181csasgcgaGfGfAfagaau 251 UfsCfsgguAfuucuucc accsgsa UfcGfcugsusuNM_001306068_1237_1255 1237 182 asgscgagGfAfAfgaaua 252UfsCfscggUfauucuuc ccgsgsa CfuCfgcususu NM_001306068_1238_1256 1238 183gscsgaggAfAfGfaauac 253 UfsCfsccgGfuauucuu cggsgsa CfcUfcgcsusuNM_001306068_1284_1302 1284 184 gsusugggAfCfGfgggu 254UfsAfscccGfaccccguC cgggsusa fcCfaacsusu NM_001306068_1290_1308 1290 185ascsggggUfCfGfggug 255 UfsCfsgaaCfcacccgaC guucsgsa fcCfcgususuNM_001306068_1294_1312 1294 186 gsgsucggGfUfGfguuc 256UfsGfscccCfgaaccacC ggggscsa fcGfaccsusu NM_001306068_1295_1313 1295 187gsuscgggUfGfGfuucg 257 UfsUfsgccCfcgaaccaC gggcsasa fcCfgacsusuNM_001306068_1315_1333 1315 188 gscsggugGfCfCfucucu 258UfsCfsgaaAfgagaggc uucsgsa CfaCfcgcsusu NM_001306068_1316_1334 1316 189csgsguggCfCfUfcucuu 259 UfsGfscgaAfagagagg ucgscsa CfcAfccgsusuNM_001306068_1317_1335 1317 190 gsgsuggcCfUfCfucuuu 260UfsCfsgcgAfaagagag cgcsgsa GfcCfaccsusu NM_001306068_1321_1339 1321 191gscscucuCfUfUfucgcg 261 UfsUfscccCfgcgaaagA gggsasa fgAfggcsusuNM_001306068_1340_1358 1340 192 csasccugGfCfUfggcua 262UfsUfsccgUfagccagcC cggsasa faGfgugsusu NM_001306068_1350_1368 1350 193gscsuacgGfAfGfgggcg 263 UfsGfsacaCfgccccucC uguscsa fgUfagcsusuNM_001306068_1351_1369 1351 194 csusacggAfGfGfggcgu 264UfsAfsgacAfcgccccuC gucsusa fcGfuagsusu NM_001306068_1539_1557 1539 195ascsgugcAfAfGfggagc 265 UfsGfscgaGfcucccuu ucgscsa GfcAfcgususuNM_001306068_1540_1558 1540 196 csgsugcaAfGfGfgagcu 266UfsAfsgcgAfgcucccu cgcsusa UfgCfacgsusu NM_001306068_1541_1559 1541 197gsusgcaaGfGfGfagcuc 267 UfsCfsagcGfagcucccU gcusgsa fuGfcacsusuNM_001306068_1610_1628 1610 198 csasccuuCfCfGfacgcu 268UfsAfsgacAfgcgucgg gucsusa AfaGfgugsusu NM_001306068_1611_1629 1611 199ascscuucCfGfAfcgcug 269 UfsUfsagaCfagcgucg ucusasa GfaAfggususuNM_001306068_1612_1630 1612 200 cscsuuccGfAfCfgcugu 270UfsCfsuagAfcagcguc cuasgsa GfgAfaggsusu NM_001306068_1613_1631 1613 201csusuccgAfCfGfcuguc 271 UfsCfscuaGfacagcguC uagsgsa fgGfaagsusuNM_001306068_1615_1633 1615 202 uscscgacGfCfUfgucua 272UfsUfsgccUfagacagc ggcsasa GfuCfggasusu NM_001306068_1616_1634 1616 203cscsgacgCfUfGfucuag 273 UfsUfsugcCfuagacag gcasasa CfgUfcggsusuNM_001306068_1619_1637 1619 204 ascsgcugUfCfUfaggca 274UfsGfsguuUfgccuaga aacscsa CfaGfcgususu NM_001306068_1632_1650 1632 205asasaccuGfGfAfuuaga 275 UfsUfsaacUfcuaauccA guusasa fgGfuuususuNM_001306068_336_354 336 206 csusuugaGfAfAfggauc 276 UfsAfsagcGfauccuucgcususa UfcAfaagsusu NM_001306068_672_690 672 207 gscscggcAfGfAfgggga277 UfsGfsagaUfccccucu ucuscsa GfcCfggcsusu NM_001306068_882_900 882 208gsgsgccaAfGfGfggugc 278 UfsGfscaaGfcaccccuU uugscsa fgGfcccsusuNM_001306068_884_902 884 209 gscscaagGfGfGfugcuu 279 UfsGfscgcAfagcacccCgcgscsa fuUfggcsusu NM_001306068_1045_1063 1045 210 asusgcaaGfGfCfauccc280 UfsCfsgccGfggaugcc ggcsgsa UfuGfcaususu vpN = vinyl phosphonate2′-MOE; upper case (N) = 2′-OH (ribo); lower case (n) = 2′-O-Me (methyl)dN = 2′-H (deoxy); Nf = 2′-F (fluoro); s = phosphorothioate backbonemodification; iB = inverted abasic

TABLE 13 SEQ ID SEQ ID NO sense/passenger_seq (5′-3′) NOantisense/guide_seq (5′-3′) 371 CTGCCTCTCCACCAGCCCA 372TGGGCTGGTGGAGAGGCAG 373 GCAGAGATGGAGAGAGGAA 374 TTCCTCTCTCCATCTCTGC 375GCGGTTTCCTCCGGGACAA 376 TTGTCCCGGAGGAAACCGC 377 GGACGACGGAGGCGTGATT 378AATCACGCCTCCGTCGTCC 379 CGGGCACCCGGAAACATGCAGGG 380TTCCCTGCATGTTTCCGGGTGCCC AA G 381 CCGGAAACATGCAGGGAAG 382CTTCCCTGCATGTTTCCGG 383 GAAATGAACGAGAGCCAGA 384 TGTGGCTCTCGTTCATTTC 385TGGCACACTCAAGACTCCCACGG 386 CTCCGTGGGAGTCTTGAGTGTGC AG CA 387CCACGGAGGTTCAGTTCCA 388 TGGAACTGAACCTCCGTGG 389 ACCACCACCACCACCACCA 390TGGTGGTGGTGGTGGTGGT 391 CGCCATTCATGAAGGGGTG 392 CACCCCTTCATGAATGGCG 393CATGAAGGGGTGGAGCCTG 394 CAGGCTCCACCCCTTCATG 395 GAGCCTGCTTTGAGCGGAA 396TTCCGCTCAAAGCAGGCTC 397 CCGAGCCTTTGAGAAGGATCGCT 398AAAGCGATCCTTCTCAAAGGCTC TT GG 399 GGCAGGGCGCCCGCGCAGG 400CCTGCGCGGGCGCCCTGCC 401 GATGATTAGTTCAGAGATA 402 TATCTCTGAACTAATCATC

Example 2. siRNA Sequences and Synthesis

All siRNA single strands were fully assembled on solid phase usingstandard phosphoramidite chemistry and purified over HPLC. Purifiedsingle strands were duplexed to get the double stranded siRNA. All thesiRNA passenger strand contains conjugation handles in differentformats, C₆—NH₂ and/or C₆—SH, one at each end of the strand. Theconjugation handle or handles were connected to the siRNA passengerstrand or siRNA guide strand via inverted abasic phosphodiester orphosphorothioate. Below are representative structures of the formatsused in the in vivo experiments.

A representative structure of siRNA with C6-NH₂ conjugation handle atthe 5′ end and C₆—SH at 3′end of the passenger strand or guide strand.

A representative structure of siRNA passenger strand or guide strandwith C₆—NH₂ conjugation handle at the 5′ end and C6-S-PEG at 3′ end.

A representative structure of siRNA passenger strand or guide strandwith C₆—NH₂ conjugation handle at the 5′ end and C₆—S-NEM at 3′ end.

A representative structure of siRNA passenger strand with C₆-N-SMCCconjugation handle at the 5′ end and C₆—S-NEM at 3′ end.

A representative structure of siRNA passenger strand or guide strandwith PEG at the 5′ end and C₆—SH at 3′ end.

A representative structure of siRNA passenger strand or guide strandwith C₆-S-NEM at the 5′ end and C₆—NH₂ conjugation handle at 3′ end.

Example 3. Conjugate Synthesis

The following structures illustrate exemplary A-X₁-B-X₂-Y (Formula I)architectures described herein.

Architecture-1: Antibody-Cys-SMCC-5′-passenger strand. This conjugatewas generated by antibody inter-chain cysteine conjugation to maleimide(SMCC) at the 5′ end of passenger strand.

Architecture-2: Antibody-Cys-SMCC-3′-Passenger strand. This conjugatewas generated by antibody inter-chain cysteine conjugation to maleimide(SMCC) at the 3′ end of passenger strand.

ASC Architecture-3: Antibody-Cys-bisMal-3′-Passenger strand. Thisconjugate was generated by antibody inter-chain cysteine conjugation tobismaleimide (bisMal)linker at the 3′ end of passenger strand.

ASC Architecture-4: A model structure of the Fab-Cys-bisMal-3′-Passengerstrand. This conjugate was generated by Fab inter-chain cysteineconjugation to bismaleimide (bisMal) linker at the 3′ end of passengerstrand.

ASC Architecture-5. A model structure of the antibody siRNA conjugatewith two different siRNAs attached to one antibody molecule. Thisconjugate was generated by conjugating a mixture of SSB and HPRT siRNAsto the reduced mAb inter-chain cysteines to bismaleimide (bisMal) linkerat the 3′ end of passenger strand of each siRNA.

ASC Architecture-6: A model structure of the antibody siRNA conjugatewith two different siRNAs attached. This conjugate was generated byconjugating a mixture of SSB and HPRT siRNAs to the reduced mAbinter-chain cysteines to maleimide (SMCC) linker at the 3′ end ofpassenger strand of each siRNA.

Example 3.1 Antibody siRNA Conjugate Synthesis Using SMCC Linker

Step 1: Antibody Interchain Disulfide Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 10mg/ml concentration. To this solution, 2 equivalents of TCEP in waterwas added and rotated for 2 hours at RT. The resultant reaction mixturewas buffer exchanged with pH 7.4 PBS containing 5 mM EDTA and added to asolution of SMCC-C6-siRNA or SMCC-C6-siRNA-C6-NHCO-PEG-XkDa (2equivalents) (X=0.5 kDa to 10 kDa) in pH 7.4 PBS containing 5 mM EDTA atRT and rotated overnight. Analysis of the reaction mixture by analyticalSAX column chromatography showed antibody siRNA conjugate along withunreacted antibody and siRNA.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1 as described in Example 3.4.Fractions containing DAR1 and DAR>2 antibody-siRNA-PEG conjugates wereseparated, concentrated and buffer exchanged with pH 7.4 PBS.

Step 3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by SEC, SAX chromatographyand SDS-PAGE. The purity of the conjugate was assessed by analyticalHPLC using either anion exchange chromatography method-2 or anionexchange chromatography method-3. Both methods are described in Example3.4. Isolated DAR1 conjugates are typically eluted at 9.0+0.3 min onanalytical SAX method and are greater than 90% pure. The typical DAR>2cysteine conjugate contains more than 85% DAR2 and less than 15% DAR3.

Example 3.2. Antibody siRNA Conjugate Synthesis Using Bis-Maleimide(BisMal) Linker

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with borax buffer (pH 8) and made up to 5mg/mi concentration. To this solution, 2 equivalents of TCEP in waterwas added and rotated for 2 hours at RT. The resultant reaction mixturewas exchanged with pH 7.4 PBS containing 5 mM EDTA and added to asolution of BisMal-C6-siRNA-C6-S-NEM (2 equivalents) in pH 7.4 PBScontaining 5 mM EDTA at RT and kept at 4° C. overnight. Analysis of thereaction mixture by analytical SAX column chromatography showed antibodysiRNA conjugate along with unreacted antibody and siRNA.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR andDAR2 antibody-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The isolated conjugates were characterized by either mass spec orSDS-PAGE. The purity of the conjugate was assessed by analytical HPLCusing either anion exchange chromatography method-2 or 3 as well as sizeexclusion chromatography method-1.

Example 3.3. Fab′ Generation from mAb and Conjugation to siRNA

Step 1: Antibody Digestion with Pepsin

Antibody was buffer exchanged with pH 4.0, 20 mM sodium acetate/aceticacid buffer and made up to 5 mg/ml concentration. Immobilized pepsin(Thermo Scientific, Prod #20343) was added and incubated for 3 hours at37° C. The reaction mixture was filtered using 30 kDa MWCO Amicon spinfilters and pH 7.4 PBS. The retentate was collected and purified usingsize exclusion chromatography to isolate F(ab′)2. The collected F(ab′)2was then reduced by 10 equivalents of TCEP and conjugated withSMCC-C₆-siRNA-PEG5 at room temperature in pH 7.4 PBS. Analysis ofreaction mixture on SAX chromatography showed Fab-siRNA conjugate alongwith unreacted Fab and siRNA-PEG.

Step 2: Purification

The crude reaction mixture was purified by AKTA explorer FPLC usinganion exchange chromatography method-1. Fractions containing DAR1 andDAR2 Fab-siRNA conjugates were separated, concentrated and bufferexchanged with pH 7.4 PBS.

Step-3: Analysis of the Purified Conjugate

The characterization and purity of the isolated conjugate was assessedby analytical HPLC using anion exchange chromatography method-2 or 3 aswell as by SEC method-1.

Example 3.4. Purification and Analytical Methods

Anion exchange chromatography method (SAX)-1.

-   -   1. Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15        cm, 13 um    -   2. Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS,        1.5 M NaCl, pH 8.0; Flow Rate: 6.0 ml/min    -   3. Gradient:

a. % A % B Column Volume b. 100 0 1.00 c. 60 40 18.00 d. 40 60 2.00 e.40 60 5.00 f. 0 100 2.00 g. 100 0 2.00

Anion Exchange Chromatography (SAX) Method-2

-   -   1. Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm    -   2. Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80%        10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 0.75 ml/min    -   3. Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 13.00 40 60f. 15.00 90 10 g. 20.00 90 10

Anion Exchange Chromatography (SAX) Method-3

-   -   1. Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm    -   2. Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80%        10 mM TRIS pH 8, 20% ethanol, 1.5 M NaCl    -   3. Flow Rate; 0.75 ml/min    -   4. Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 23.00 40 60f. 25.00 90 10 g. 30.00 90 10

Size Exclusion Chromatography (SEC) Method-1

-   -   1. Column: TOSOH Biosciences, TSKgelG3000SW XL, 7.8×300 mm, 5 μM    -   2. Mobile phase: 150 mM phosphate buffer    -   3. Flow Rate: 1.0 ml/min for 15 mins

Example 3.5. Antibody siRNA Conjugate Synthesis Using Bis-Maleimide(BisMal) Linker

Step 1: Antibody Reduction with TCEP

Antibody was buffer exchanged with 25 mM borate buffer (pH 8) with 1 mMDTPA and made up to 10 mg/ml concentration. To this solution, 4equivalents of TCEP in the same borate buffer were added and incubatedfor 2 hours at 37° C. The resultant reaction mixture was combined with asolution of BisMal-siRNA (1.25 equivalents) in pH 6.0 10 mM acetatebuffer at RT and kept at 4° C. overnight. Analysis of the reactionmixture by analytical SAX column chromatography showed antibody siRNAconjugate along with unreacted antibody and siRNA. The reaction mixturewas treated with 10 EQ of N-ethylmaleimide (in DMSO at 10 mg/mL) to capany remaining free cysteine residues.

Step 2: Purification

The crude reaction mixture was purified by AKTA Pure FPLC using anionexchange chromatography (SAX) method-1. Fractions containing DAR1 andDAR2 antibody-siRNA conjugates were isolated, concentrated and bufferexchanged with pH 7.4 PBS.

Anion exchange chromatography method (SAX)-1.

Column: Tosoh Bioscience, TSKGel SuperQ-5PW, 21.5 mm ID×15 cm, 13 um

Solvent A: 20 mM TRIS buffer, pH 8.0; Solvent B: 20 mM TRIS, 1.5 M NaCl,pH 8.0; Flow Rate: 6.0 ml/min

Gradient:

a. % A % B Column Volume b. 100 0 1 c. 81 19 0.5 d. 50 50 13 e. 40 600.5 f. 0 100 0.5 g. 100 0 2

Anion Exchange Chromatography (SAX) Method-2

Column: Thermo Scientific, ProPac™ SAX-10, Bio LC™, 4×250 mm

Solvent A: 80% 10 mM TRIS pH 8, 20% ethanol; Solvent B: 80% 10 mM TRISpH 8, 20% ethanol, 1.5 M NaCl; Flow Rate: 0.75 ml/min

Gradient:

a. Time % A % B b. 0.0 90 10 c. 3.00 90 10 d. 11.00 40 60 e. 14.00 40 60f. 15.00 20 80 g. 16.00 90 10 h. 20.00 90 10

Example 4. Expression Profile of DUX4 in Myonuclei

Myotubes derived from a healthy individual and FSHD patient wereevaluated for DUX4 expression. As shown in FIG. 4 , myotubes wereimmunostained to detect DUX4 expression. Muscle cells' nuclei andcytoplasm were immunostained by labeling DAPI(4′,6-diamidino-2-phenylindole) and troponin T. As shown, DUX4expression could be detected in low and sporadic manner—in less than 1%of myonuclei—indicating that detecting of DUX4 expression directly fromthe cell can be challenging in determining the effect of DUX4 siRNAactivity.

Example 5. Expression Profiles of DUX4-Dependent Marker Genes in FSHDDonor Muscle Cells with DUX4 siRNA Treatment

Two DUX4 siRNAs (siDUX4-1 and siDUX4-4 are disclosed in Geng L N et al.Dev. Cell, 2012) were used to treat the diseased muscle cells (FSHDdonor muscle cells), and RNA expression level of five DUX4-dependentbiomarker genes were quantitated, as shown in bar graphs in FIG. 5 . Asshown, both siDUX4-1 and siDUX4-4 substantially reduced the expressionof MBD3L2. TRIM43, PRAMEF1, ZSCAN4, and LEUTX compared to the baseline(100%). More specifically, siDUX4-4 reduced the expression of MBD3L2,TRIM43, PRAMEF1, ZSCAN4, and LEUTX at least ˜75% compared to thebaseline (100%). DUX4-target genes as biomarkers were sensitive tomeasure siRNA-mediated downregulation of DUX4.

Example 6. DUX4 siRNA-Mediated Reduction of 5 DUX4-Target Biomarker GeneExpressions and a FSHD Composite of DUX4-Target Biomarker Genes inCultured FSHD Primary Myotubes

FSHD1 patient-derived primary myoblasts (MB06) were used to validate theFSHD composite as a reliable surrogate biomarker to assess DUX4 siRNAactivity with the published 2 DUX4 siRNAs, siDUX4-1 and siDUX4-4 (Geng LN et al, Dev. Cell, 2012) in a dose dependent concentration. FSHDprimary myoblasts (MB06 (FSHD1)) were grown in recommended media. Priorto seeding, 96-well tissue culture plates (Costar) were coated with 50μL of 1% Matrigel per well for at least 2 hours at 37° C. and washed 2×with PBS. After coating, myoblasts were seeded in quadruplicate at 4000cells/well without antibiotics and maintained 24 h prior totransfection. On the day of transfection, the DUX4-4 siRNA wasformulated with commercially available transfection reagentLipofectamine RNAiMAX (Life Technologies) and OptiMEM (LifeTechnologies) according to the manufacturer's “forward transfection”instructions. DUX4-4 siRNA was synthesized by Integrated DNATechnologies (IDT). Myoblasts were transfected with DUX4-4 siRNA at ahigh concentration of 25 nM with 9-fold serial dilutions, 24 hours posttransfection, myogenic differentiation was induced with 15%KOSR-containing differentiation medium. Myotubes were collected inTrizol 4 days after inducing differentiation and stored at −80′C untilprocessing. RNA isolation was performed using Direct-zol-96 RNAisolation kit (Zymo) according to the manufacturer's instructions,100-500 ng of purified RNA was converted to cDNA using High-CapacitycDNA Reverse Transcription Kit (Applied Biosystems) using SimpliAmpThermal Cycler (Applied Biosystems). cDNA was analyzed by qPCR usingTaqMan Fast Universal Master Mix II (Thermo Fisher) and TaqMan probes(Thermo Fisher) in duplicates, using QuantStudio 6 or 7 Flex Real-TimePCR instruments (Applied Biosystems). Data were analyzed byQuantStudio-Real-Time PCR Software v1.3 (Applied Biosystems). Theexpression levels of 5 DUX4-target genes were evaluated: MBD3L2, ZSCAN4,LEUTX, KHDCIL, and TRIM43. DUX4-target gene expression was normalized toa composite of two reference genes: AHSA1 and RPL27. The percentage oftarget mRNA expression was determined relative to mock treated cells byusing the 2-method (Livak and Schmittgen, Methods 2001). FIG. 6A-B showDUX4 siRNA-mediated reduction of 5 DUX4-target biomarker geneexpressions in cultured FSHD primary myotubes. DUX4 siRNA reduced theexpression of 5 individual DUX4-target biomarker genes (MBD3L2. ZSCAN4,LEUTX, KHDC1L. and TRIM43) (FIG. 6A) or as a FSHD composite of the 5DUX4-target biomarker genes (FIG. 6B) in cultured FSHD primary myotubes.

Example 7. DUX4 siRNA Library Screening—First Round at 2 Concentrations(10 and 0.5 nM) in MB02 and MB06

In this example, 70 DUX4 siRNAs were screened for their activity in twoFSHD primary myoblast cell lines (MB02 (FSHD1) and MB06 (FSHD1)) toidentify more desirable siRNA candidates. Cells were plated at a densityof 4,000 cells/well (MW96) and then transfected in four replicates withDUX4 siRNAs, as well as a tool siRNA as control. Transfection wasperformed 24 hours after plating at 10 nM concentration. Myogenicdifferentiation was induced 2 days after plating (24 hours aftertransfection) with 15% KOSR (in DMEMF-12) medium. Samples were harvested3 or 4 days after inducing differentiation, depending on the cell line.DUX4-downstream target gene expression was evaluated by RT-qPCR(normalized to a composite AHSA1 and RPL27 housekeeping gene expressionvalue). Data shown in this example are represented as mean FSHDComposite −/+SEM. N=4.

FIG. 7 shows a bar graph of assessment of myotubes differentiation andviability at 10 nM concentration of siDUX4 by monitoring ACTA expressionin MB U2 (FSHD1) and MB 6 (FSHDC1) cell lines.

FIG. 8 shows a bar graph of screening for activity at 10 nMConcentration of 70 DUX4 siRNAs by measuring FSHD composite expression(Calculations of the FSHD composite: Dct=(Av. ct 4 DUX4 targetgenes)−(Av. ct 2 HKGs), DDct=Dct (siDUX4)−Dct (mock),Composite=2{circumflex over ( )}−DDct*100(%)).

FIG. 9 shows a bar graph of screening for activity at 0.5 nMConcentration of 70 DUX4 siRNAs by measuring FSHD composite expression.As used herein. FSHD Composite is made of 4 DUX4-target genes. MBD3L2ZSCAN4, LEUTX, KHDC 1L. As shown in Table 15, downregulation of FSHDcomposite expression is well correlated with dow-nregulation ofindividual genes in the composite when normalized to HKG composite(AHSA1, RPL27) (n=4), indicating that the effective downregulation ofFSHD composite expression is a good indicator to DUX4 siRNA potency.

TABLE 15 MB02 FSHD siRNA Composite KHDC1L LEUTX MDB3L2 ZSCAN4 AHSA1RPL27 11 31.75 32.33 27.01 47.47 30.27 109.58 91.38 57 25.85 22.62 27.7042.22 20.85 97.29 103.31 58 9.78 12.44 13.10 5.88 7.11 97.37 103.29 5943.49 44.17 43.50 45.55 45.97 105.40 94.78 60 58.45 59.45 55.13 69.7557.17 94.97 105.61 61 6.34 7.32 2.83 11.18 9.93 90.50 113.77 62 15.4418.56 12.97 25.33 12.82 100.38 102.07 63 13.73 29.72 11.40 14.50 10.0785.09 118.35 77 37.74 32.95 30.95 47.38 45.61 94.54 105.59 78 72.9759.09 58.98 102.78 83.09 100.39 100.53 79 19.50 12.62 19.45 34.83 24.75108.94 95.53 99 22.26 16.41 19.77 35.04 25.06 92.52 109.14 102 48.1739.31 49.89 65.85 45.69 101.19 100.20 137 17.49 14.10 11.02 25.26 31.3793.65 107.00 160 201.88 205.52 215.56 241.34 167.58 97.09 103.95 16261.62 51.74 58.79 91.54 57.24 105.98 93.58 163 122.38 115.49 111.83111.99 154.49 88.53 113.03 231 91.73 95.05 89.58 106.78 91.32 97.74102.50 232 23.56 25.81 22.38 33.70 17.65 102.82 98.35 274 14.70 12.587.62 26.42 21.88 95.39 106.34 276 355.14 295.05 459.25 349.12 397.99104.97 96.57 277 37.91 43.98 41.85 40.48 38.05 108.35 94.03 285 86.15108.25 75.54 78.48 98.14 88.18 114.79 287 339.10 407.15 355.95 331.14282.13 99.11 101.55 292 74.54 55.24 70.23 99.22 71.01 99.83 100.24 293103.15 93.51 103.54 114.07 115.31 95.28 104.03 294 104.91 113.05 114.54112.24 92.48 92.28 108.55 335 12.62 12.47 13.75 15.54 14.57 100.38100.54 289 101.16 143.98 74.83 103.37 105.53 91.50 110.54 524 10.70 7.905.81 18.87 20.05 102.71 97.55 525 41.37 44.14 45.56 39.27 41.85 100.52100.49 672 1105.70 976.91 1558.44 1316.38 778.03 102.53 97.86 679 126.54165.55 124.45 112.83 118.55 84.77 118.50 704 93.68 93.39 94.12 118.5485.98 100.74 100.94 705 52.22 52.53 39.24 70.91 49.46 100.96 99.96 MB06FSHD siRNA Composite KHDC1L LEUTX MBD3L2 ZSCAN4 AHSA1 RPL27 11 16.098.38 11.09 23.82 33.31 114.05 90.97 57 14.22 12.15 10.51 18.81 19.5489.28 115.02 58 4.10 2.57 2.37 6.05 11.34 101.9 105.55 59 28.22 22.4829.71 33.29 32.50 117.04 88.15 60 140.69 122.45 142.62 155.90 150.70116.93 4.15 61 1.73 1.57 1.08 1.78 4.58 98.5 114.58 62 20.45 10.45 15.6337.90 32.54 101.13 102.58 63 17.87 21.42 13.53 24.40 16.06 83.35 122.8477 75.08 53.88 58.53 78.07 112.37 111.85 93.58 78 106.34 79.97 85.79123.98 154.02 103.95 101.34 79 5.72 7.19 3.78 5.70 8.23 99.32 104.82 9935.72 25.35 26.35 40.43 63.61 99.65 104.80 102 44.10 57.01 39.54 42.8340.52 90.72 119.87 137 17.65 12.4 12.00 21.07 33.17 98.95 105.09 160300.78 306.57 304.41 294.44 312.89 108.55 100.20 162 45.68 29.38 37.7055.89 73.25 105.10 97.57 163 79.05 63.62 72.05 62.17 144.47 95.43 109.30231 337.97 283.17 379.63 352.81 354.24 115.95 92.45 232 12.55 11.16 9.3315.35 17.15 92.25 109.99 274 34.53 26.94 29.25 37.69 50.61 103.83 99.85276 319.52 337.69 319.51 228.18 445.11 99.52 110.28 277 18.43 17.3816.75 19.54 22.05 100.75 102.23 285 65.05 53.72 56.52 72.14 71.55 102.87103.35 287 239.05 253.18 231.21 228.18 257.25 101.75 102.15 292 141.85143.88 135.19 159.14 135.01 103.00 105.57 293 55.65 40.83 48.59 71.5370.82 103.57 100.30 294 147.19 163.93 152.23 129.50 154.54 104.25 104.91335 1.77 1.85 0.74 2.24 3.69 105.87 100.27 289 87.13 87.19 61.30 80.98139.82 97.28 110.00 524 11.57 8.50 7.01 13.83 22.05 125.94 84.71 525147.51 131.00 168.52 148.43 149.22 115.00 89.05 672 875.88 932.45 950.25747.57 916.32 95.33 105.59 679 111.13 89.15 108.25 119.11 139.93 108.5999.35 704 382.40 345.98 392.55 385.90 415.94 110.04 98.08 705 37.8540.55 22.82 47.40 50.42 95.14 108.51 MB02 FSHD siRNA Composite KHDC1LLEUTX MDB3L2 ZSCAN4 AHSA1 RPL27 708 19.24 27.41 17.79 21.23 14.34 105.7495.27 882 65.40 41.13 61.93 118.03 64.59 108.83 93.30 884 34.58 28.8225.19 51.92 42.17 106.64 95.66 893 15.06 12.73 13.13 23.42 14.15 80.16124.98 1045 33.86 28.20 32.38 42.01 42.10 102.03 99.58 1132 3.20 4.191.89 4.26 4.64 92.42 110.66 1134 41.73 31.74 27.87 62.98 58.37 97.67103.39 1158 7.01 6.51 4.48 13.33 9.47 91.29 109.76 1159 226.21 152.73154.74 292.83 401.40 82.15 126.63 1163 11.52 15.00 8.01 15.75 10.5594.55 106.71 1236 4.22 3.32 1.94 8.35 8.47 82.34 121.73 1237 32.87 31.0225.17 43.61 38.79 97.35 102.99 1238 24.43 25.07 19.03 35.16 24.46 105.0295.44 1284 90.11 83.12 84.62 106.45 100.99 98.75 106.13 1290 104.3697.53 102.02 118.57 109.73 100.22 101.46 1294 108.34 91.38 115.59 177.2478.03 114.67 88.47 1295 60.35 60.16 43.80 96.21 55.94 69.18 145.68 131592.84 85.02 96.47 111.78 83.44 107.21 93.35 1316 82.53 68.66 62.91154.83 71.10 97.30 103.65 1317 104.92 105.43 101.20 112.07 112.09 97.95105.07 1321 23.72 20.71 13.75 44.87 29.79 87.40 115.65 1340 13.06 14.5514.52 14.04 12.12 76.27 131.90 1350 207.49 155.67 224.45 254.19 222.09103.71 96.95 1351 2217.65 1074.92 2732.04 3210.96 2752.93 135.49 75.801539 85.20 76.82 87.54 121.01 73.71 98.65 102.96 1540 27.76 27.63 21.7439.45 27.56 99.84 100.4 1541 27.45 23.47 21.75 36.77 33.40 94.71 106.551610 28.54 14.04 21.45 56.20 41.60 102.33 98.02 1611 31.12 23.09 26.4259.28 27.96 96.38 104.00 1612 55.31 46.84 46.05 91.02 53.05 104.00 97.071613 5.18 6.53 1.37 8.42 9.92 96.50 106.13 1615 39.81 36.59 35.01 62.9435.85 103.77 97.50 1616 31.13 28.01 32.41 33.99 31.93 94.67 106.96 161916.06 11.42 7.09 38.40 25.85 96.42 104.14 1632 17.02 21.17 23.00 26.398.73 109.10 92.79 MB06 FSHD siRNA Composite KHDC1L LEUTX MBD3L2 ZSCAN4AHSA1 RPL27 708 13.86 12.61 8.91 21.99 16.45 110.19 98.33 882 95.0351.51 92.26 137.90 135.15 126.73 85.08 884 25.97 22.07 25.24 23.84 38.18121.69 88.93 893 25.57 22.57 17.56 18.86 61.47 74.41 151.83 1045 72.2055.02 62.24 73.95 110.04 112.74 93.00 1132 2.17 1.38 1.22 2.33 4.89110.80 95.16 1134 165.18 124.57 138.47 238.72 194.16 120.19 96.87 11588.01 6.27 3.33 14.17 16.01 103.69 100.97 1159 525.79 246.31 419.47961.82 840.11 109.42 94.92 1163 0.62 0.71 0.24 1.16 1.49 114.57 93.451236 1.85 1.56 0.70 2.59 4.72 83.65 125.55 1237 18.02 15.99 12.24 19.8328.35 86.15 127.14 1238 29.53 23.79 22.54 35.10 43.41 106.57 101.82 128426.55 26.17 21.71 21.91 47.85 105.06 107.66 1290 335.73 277.05 410.94346.25 336.63 129.53 81.28 1294 210.94 212.54 213.33 192.41 237.66104.07 108.48 1295 26.36 19.44 20.39 39.85 33.69 80.55 134.12 1315132.47 131.69 120.09 124.25 162.31 102.73 107.96 1316 170.76 141.70168.39 207.31 175.30 116.86 93.87 1317 155.72 136.41 146.45 192.41163.46 116.80 89.59 1321 30.38 22.32 20.48 43.92 44.59 91.46 115.30 134012.24 16.78 10.49 8.35 17.48 80.76 135.39 1350 108.59 73.33 116.32128.51 130.28 111.71 94.61 1351 556.04 409.02 572.93 604.80 690.63124.43 89.12 1539 174.37 162.38 150.76 180.05 217.16 105.72 100.85 15406.96 10.19 3.38 8.01 9.77 95.47 111.96 1541 19.68 13.08 13.65 27.7134.20 104.27 100.39 1610 26.04 14.84 20.65 28.45 59.48 111.99 98.08 1611148.02 126.75 169.36 178.77 128.62 115.44 90.38 1612 166.82 149.98188.56 161.52 174.12 123.98 87.12 1613 13.55 10.97 6.06 22.26 24.00110.12 94.54 1615 15.05 10.29 8.06 20.20 34.44 98.28 107.83 1616 24.9515.42 15.50 31.87 59.94 104.72 99.75 1619 19.69 15.64 10.91 33.63 30.66103.27 106.14 1632 4.83 6.04 2.20 7.24 6.80 101.92 104.27

As shown in FIG. 10 , from the DUX4 siRNA library with 70 siRNAcandidates 37 candidates were eliminated that have lower than 70% KD inat least one cell lines at 10 nM. Then, 3 additional siRNA candidateswere eliminated that resulted in ACTA1 expression less than 70% in bothcell lines. Then, 2 additional siRNA candidates with null activity (<10%KD) at 0.5 nM in both cell lines were eliminated, resulting in total 28siRNA candidates left for the next screening process. Table 16 listssuch selected 28 DUX4 siRNAs and the downregulation of FSHD compositeexpression in two cell lines at the concentration of 10 nM and 0.5 nM ofthe selected 28 DUX4 siRNAs (also shown as bar graphs in FIGS. 11A and11B).

TABLE 16 FSHD Composite (% Mock) MB02- MB02- MB06- MB06- siRNA 10 nM 0.5nM 10 nM 0.5 nM 11 31.76 51.87 16.09 76.24 57 25.86 64.82 14.22 38.21 589.78 40.95 4.10 88.52 61 6.34 34.34 1.73 48.20 62 15.44 45.95 20.4685.52 63 13.73 52.64 17.87 79.18 79 19.50 56.01 5.72 71.70 99 22.2663.90 35.72 91.67 137 17.49 39.13 17.66 71.37 277 37.91 76.29 18.43107.75 336 12.62 31.51 1.77 43.22 524 10.70 53.98 11.57 73.00 708 19.2473.21 13.86 72.29 1132 3.20 59.37 2.17 66.00 1158 7.01 30.58 8.01 54401163 11.52 48.46 0.62 37.70 1236 4.22 25.00 1.85 16.55 1237 32.87 99.2518.02 47.65 1238 24.43 60.92 29.53 82.40 1340 13.06 38.81 12.24 47.631540 27.76 17.63 6.96 17.57 1541 27.45 34.55 19.68 70.70 1610 28.5450.65 26.04 76.91 1613 5.18 18.30 13.55 39.25 1615 39.81 45.49 15.0550.57 1616 31.13 37.63 24.95 32.25 1619 16.06 42.95 19.69 49.95 163217.02 20.07 4.83 37.10

Databases of human polymorphisms are not reliable in repeats and it ispossible that polymorphic positions may be missed. Thus, in this roundof selection, siRNAs that show poor performance in one of two myoblastcell lines were eliminated for a purpose of validating that selectedDUX4 siRNAs are active in a variety of FSHD-patient derived myoblasts.FIG. 12 shows distribution of 70 siRNAs by their efficacies ofdownregulating FSHD composite in two myoblast cell lines. KD correlationanalysis identified −10% of DUX4 siRNAs that worked only in one of thetwo FSHD primary myoblast cell lines used in the full library screening.

Example 8. DUX4 siRNA Library Screening—Second Round at 10 nM in FourDifferent Patient-Derived Primary Myoblast Cell Lines

In this example, a total of 9 high quality FSHD patient-derived primarymyoblast cell lines (6 FSHD1, 3 FSHD2) were used. Conditions that allowreliable detection of DUX4 target gene expression in FSHD myotubesin-house were established by culturing the cells in Differentiationmedium (15% KOSR in DMEM/F-12). Time-point was selected specifically foreach cell line. All 9 FSHD cell lines showed a concentration-dependentresponse to a tool DUX4 siRNA.

FIG. 13 shows a bar graph of ACTA1 expression levels in four differentpatient-derived primary myoblast cell lines (MB01, MB05, MB1 1, MB12).Most of the top 28 DUX4 siRNAs did not effect ACTA1 expression levels inthese cell lines, while a few siRNAs showed more than 30% reduction inACTA1 expression levels in the additional 4 FSHD primary cell linestested (8 in MB1 1, 3 in MB05, 3 in MB12, and none in MB01). Inaddition, as shown in FIG. 14 , the top 28 DUX4 siRNAs showed activityin all four additional FSHD primary cell lines. Several siRNAs showedmore than 75% KD in three lines: MB05, MB11, MB12. KD levels wereoverall lower in MB01. FIG. 15 shows the top 28 DUX4 siRNAs' activity at10 nM concentration in all FSHD cell lines (MB01, MB02, MB05, MB06, MB11, MB12).

Top 14 DUX4 siRNAs were selected from top 28 DUX4 siRNAs. Selection ofsuch top 14 DUX4 siRNAs were i) siRNAs that showed no or minimal celltoxicity (visual identification and evaluation of ACTA 1 expressionlevels), and ii) siRNAs that displayed the best activity at both 10 nMand 0.5 nM concentrations. Table 17 lists the top 14 DUX4 siRNAs anddownregulation of FSHD composite expression in six primary FSHD celllines (MB01, MB02, MB05, MB06, MB1 1. MB12) at the concentration of 10nM and 0.5 nM of the selected 14 DUX4 siRNAs, and downregulation ofACTA1 expression in six primary FSHD cell lines at the concentration of10 nM. Table 18 lists the top 14 DUX4 siRNAs and downregulation of FSHDcomposite expression in all 9 FSHD primary myotubes.

TABLE 17 FSHD Composite (% Mock) 10 nM 0.5 nM siRNA MB01 MB02 MB05 MB06MB11 MB12 MB02 MB06 11 43.10 31.76 15.02 16.09 2.66 13.06 51.87 76.24 5760.89 25.86 16.05 14.22 29.01 43.75 64.82 38.21 58 47.60 9.78 8.74 4.105.00 25.30 40.95 88.52 61 25.54 6.34 6.82 1.73 0.63 4.11 34.34 48.20 13771.58 17.49 26.43 17.66 7.90 18.69 39.13 71.37 336 36.05 12.62 15.531.77 0.96 6.47 31.51 43.22 1132 68.25 3.20 19.20 2.17 13.69 25.30 59.3766.00 1340 56.58 13.06 21.04 12.24 18.09 22.73 38.81 47.63 1540 73.6927.76 32.13 6.96 3.91 25.71 17.63 17.57 1613 39.26 5.18 16.04 13.5514.70 19.28 18.30 39.25 1615 60.45 39.81 17.82 15.05 16.36 20.41 45.4950.57 1616 81.11 31.13 46.69 29.45 8.98 24.15 37.63 32.25 1619 82.8316.06 24.32 19.69 12.41 28.99 42.95 49.95 1632 54.74 17.02 11.74 4.834.99 12.45 20.07 37.10 ACTA 1 (% Mock) 10 nM siRNA MB01 MB02 MB05 MB06MB11 MB12 11 182.43 149.99 114.72 134.18 74.18 123.86 57 134.81 133.8777.42 136.08 101.53 122.19 58 164.25 152.35 119.96 159.71 89.04 103.7461 161.47 112.20 91.46 95.20 73.28 85.37 137 175.58 159.65 113.40 140.4085.80 114.48 336 113.53 92.45 99.88 123.50 159.92 96.08 1132 162.91132.10 98.43 59.31 74.15 125.51 1340 237.27 204.96 176.84 346.51 167.31134.73 1540 144.35 114.80 152.76 139.56 103.36 126.51 1613 155.21 119.51107.74 164.23 125.38 122.92 1615 127.69 100.20 125.61 125.54 76.09 97.171616 115.10 74.43 89.58 77.81 52.83 80.71 1619 147.16 205.75 165.69208.77 203.81 172.31 1632 151.01 135.47 147.94 162.22 101.99 157.04

TABLE 18 FSHD Composite (% Mock) 10 nM 0.5 nM siRNA MB01 MB02 MB03 MB04MB05 MB06 MB10 MB11 MB12 MB02 MB06 11 43.10 31.76 22.99 5.61 15.02 16.0919.78 2.66 13.06 51.87 76.24 57 60.89 25.86 35.58 81.33 16.05 14.2233.53 29.01 43.75 64.82 38.21 58 47.60 9.78 17.90 0.72 8.74 4.10 22.295.00 25.30 40.95 88.52 61 25.54 6.34 12.59 0.59 6.82 1.73 16.50 0.634.11 34.34 48.20 137 71.58 17.49 25.94 0.37 26.43 17.66 26.87 7.90 18.6939.13 71.37 336 36.05 12.62 15.99 0.13 15.53 1.77 11.35 0.96 6.47 31.5143.22 1132 68.25 3.20 15.18 8.72 19.20 2.17 34.72 13.69 25.30 59.3766.00 1340 56.58 13.06 41.13 13.67 21.04 12.24 35.26 18.09 22.73 38.8147.63 1540 73.69 27.76 39.02 6.94 32.13 6.96 27.94 3.910 25.71 17.6317.57 1613 39.26 5.18 13.19 76.57 16.04 13.55 84.76 14.70 19.28 18.3039.25 1615 60.45 39.81 34.04 12.48 17.82 15.05 41.32 16.36 20.41 45.4950.57 1616 81.11 31.13 36.27 13.99 46.99 24.95 72.30 8.98 24.15 37.6332.25 1619 82.83 16.06 40.97 3.88 24.32 19.69 41.16 12.41 28.99 42.9549.95 1632 54.74 17.02 19.15 4.02 11.74 4.83 28.31 4.99 12.45 20.0737.10 ACTA 1 (% Mock) 10 nM siRNA MB01 MB02 MB03 MB04 MB05 MB06 MB10MB11 MB12 11 182.43 149.99 94.53 252.83 114.72 134.18 113.25 74.18123.86 57 134.81 133.87 135.56 83.84 77.42 136.08 104.46 101.53 122.1958 164.25 152.35 201.75 146.25 119.96 159.71 82.38 89.04 103.74 61161.47 112.20 170.49 97.32 91.46 95.20 108.30 73.28 85.37 137 175.58159.65 92.10 197.01 113.40 140.40 71.16 85.80 114.48 336 113.53 92.4599.01 476.86 99.88 123.50 93.80 159.92 96.08 1132 162.91 132.10 149.30109.39 98.43 59.31 103.20 74.15 125.51 1340 237.27 204.96 182.92 279.19176.84 346.51 160.47 167.31 134.73 1540 144.35 114.80 92.99 101.69152.76 139.56 56.11 103.36 126.51 1613 155.21 119.51 160.42 289.09107.74 164.23 102.70 125.38 122.92 1615 127.69 100.20 97.77 146.44125.61 125. 45.03 76.09 97.17 1616 115.10 74.43 74.24 84.55 89.58 77.8146.80 52.83 80.71 1619 147.16 205.75 155.05 967.98 165.69 208.77 88.92203.81 172.31 1632 151.01 135.47 113.22 531.53 147.94 162.22 155.60101.99 157.04

Example 9. Top 14 DUX4 siRNAs Evaluated for Potency in ConcentrationResponse in MB02, MB05 and MB06

The goal of the experiments in this example is to select 8 siRNAs withbest Emax and potency for off-target analysis. Three FSHD primarymyoblast cell lines (MB02, MB05, M1B06) were used. Cells were plated ata density of 4,000 cells/well (MW96) and transfected in quadruplicateswith selected 14 DUX4 siRNAs. Transfection was performed 24 hours afterplating. Myogenic differentiation was induced 2 days after plating (24hours after transfection) with 15% KOSR (in DMEM/F-12) medium. Sampleswere harvested 3 or 4 days after inducing differentiation, depending onthe cell line. DUX4-downstream target gene expression was evaluated byRT-qPCR (normalized to a composite AHSA1 and RPL27 housekeeping geneexpression value). Data represented as mean−/+SEM. N=4.

FIGS. 16A-C show concentration response of 14 selected DUX4 siRNA inthree FSHD patient-derived primary myotubes—MB02, MB05, MB06,respectively. DUX-4-target gene expression was reduced more than 75% bymost of the top 14 DUX4 siRNAs in three FSHD patient-derived primarymyoblast lines. It was also observed that differences on potency amongsiRNAs ranged between 60 to 100-fold depending on the cell lines. Tables19-22 show potency of DUX4 siRNAs evaluated in three FSHDpatient-derived primary myotubes based on FSHD composite.

TABLE 19 MB02 siRNA Max KD (%)* IC50 (nM) 11 95.54 1.506 57 90.06 0.91858 100.00 1.969 61 100.00 0.639 137 93.30 1.903 336 83.74 0.110 1132100.00 2.830 1340 83.69 2.137 1540 66.47 0.047 1613 95.09 0.259 161576.01 0.166 1616 88.05 0.372 1619 87.10 0.306 1632 91.52 0.082

TABLE 20 MB05 siRNA Max KD (%)* IC50 (nM) 11 83.78 0.055 57 86.86 0.87058 89.08 0.375 61 95.24 0.127 137 86.13 0.673 336 94.35 0.127 1132100.00 2.666 1340 79.53 0.309 1540 91.33 0.075 1613 96.23 0.118 161582.43 0.091 1616 81.99 0.130 1619 78.60 0.091 1632 87.59 0.027

TABLE 21 MB06 siRNA Max KD (%)* IC50 (nM) 11 90.75 1.923 57 68.18 0.05458 98.45 1.374 61 100.00 0.665 137 87.25 2.018 336 98.53 0.359 1132100.00 4.482 1340 100.00 1.090 1540 92.71 0.145 1613 84.07 0.070 161580.01 0.457 1616 67.81 0.051 1619 71.83 0.299 1632 93.15 0.243

TABLE 22 All lines together Av. Max KD Av. IC50 siRNA (%)* (nM) 11 90.021.161 57 81.70 0.614 58 95.84 1.239 61 98.41 0.477 137 88.89 1.531 33692.21 0.199 1132 100.00 3.326 1340 87.74 1.179 1540 83.50 0.089 161391.80 0.149 1615 79.48 0.238 1616 79.28 0.184 1619 79.18 0.232 163290.76 0.117

Example 11. Antibody-DUX4 siRNA Conjugates (DUX4-AOCs) MediatedReduction of FSHD Composite (Composite of DUX4-Target Biomarker Genes)Expression in Cultured FSHD Primary Myotubes

The in vitro concentration-response potency and maximum efficacy of 16DUX4-AOCs (8 vpUq AOCs or 8 Non-VP AOCs) were assessed in FSHD1patient-derived primary myotubes (MB06). The guide strand of the DUX4siRNA of the AOCs was either with vinylphophonate at the 5′ of thestrand (vpUq) or without vinylphosphonate at the 5′ end of the strand(Non-VP).

The human IgG1 antibody against human TfR1 was expressed in CHO stablepools, created by transfecting the CHOKiSV GS-KO host cell line with adouble gene vector. The antibody was captured from cell culturesupernatant using protein A affinity chromatography. The resultantantibody was further purified using hydrophobic interactionchromatography (to reduce aggregates) and anion exchange chromatography(to reduce host cell DNA & endotoxin). The final antibody was bufferexchanged into either PBS or 50 mM sodium citrate buffer. pH 6.5 at aconcentration of 20 mg/mL. The purity of the antibody was assessed bysize exclusion chromatography.

The guide and fully complementary RNA passenger strands were assembledon solid phase using standard phospharamidite chemistry and purifiedover HPLC. Purified single strands were duplexed to obtain doublestranded siRNA. The guide strand was produced with a vinylphosphonatemodified nucleotide structures at the 5′end. The passenger strand had aconjugation handle on the 5′ end via a phosphorothioate-invertedabasic-phosphodiester linker.

Antibody oligonucleotide conjugates (AOCs) were generated using a randomcysteine conjugation method. The interchain disulfide bonds of theantibody were partially reduced with TCEP prior to conjugation with amaleimide linker-siRNA. The reaction mixture was purified using stronganion exchange chromatography to ensure a drug-antibody ratio (DAR)equal to 1 (i.e., one siRNA molecule per one antibody molecule).Collected AOC fractions were concentrated, buffer exchanged into PBS andsterile filtered using a 0.2 μm filter. The purity of AOCs was assessedusing strong anion exchange chromatography, size exclusionchromatography, and SDS-PAGE.

FSHD primary myoblasts (MB06 (FSHD1)) were grown in recommended media.Prior to seeding, 96-well tissue culture plates (Costar) were coatedwith 50 μL of 1% Matrigel per well for at least 2 hours at 37° C. andwashed 2× with PBS. After coating, myoblasts were seeded inquadruplicate at 4000 cells/well. 2 days after plating, myogenicdifferentiation was induced with 15% KOSR-containing differentiationmedium. 24 hours after inducing differentiation, siDUX4-AOCs were addedin the medium at a high concentration of 100 nM with 10-fold serialdilutions. Untreated cells were maintained as a relative control. After3 days of incubation with DUX4-AOCs, myotubes were collected in Trizoland stored at −80° C. until processing. RNA isolation was performedusing Direct-zol-96 RNA isolation kit (Zymo) according to themanufacturer's instructions. 100-500 ng of purified RNA was converted tocDNA using High-Capacity cDNA Reverse Transcription Kit (AppliedBiosystems) using SimpliAmp Thermal Cycler (Applied Biosystems). cDNAwas analyzed by qPCR using TaqMan Fast Universal Master Mix II (ThermoFisher) and TaqMan probes (Thermo Fisher) in duplicates, usingQuantStudio 6 or 7 Flex Real-Time PCR instruments (Applied Biosystems).Data were analyzed by QuantStudio™ Real-Time PCR Software v1.3 (AppliedBiosystems). DUX4-target gene expression levels were evaluated bycalculating the FSHD Composite score, which integrated the expressionlevels of 4 DUX4-target genes (MBD3L2, ZSCAN4, LEUTX, and KHDC L)normalized to two reference genes (AHSA1 and RPL27).

FIGS. 18A-B show the dose response curve of DUX4-AOCs mediated reductionof DUX4-target biomarker gene expressions in cultured FSHD primarymyotubes. Most DUX4-AOCs with vinylphosphonate (FIG. 18A) and severalDUX4-AOCs without vinylphosphonate (FIG. 18B) reduced the expression ofthe FSHD composite of 4 DUX4-target biomarker genes (MBD3L2, ZSCAN4,LEUTX, and KHDC1L) in FSHD1 patient-derived primary myotubes. Overall,DUX4-AOCs reduced the expression of DUX4-target biomarker geneexpressions and the presence of vinylphosphonate on the DUX4 siRNA inthe AOCs facilitated the reduction of the DUX4-target biomarker geneexpressions.

Example 12. Malat1-siRNA AOC Mediated In Vivo Reduction of NuclearLocalized Inc-RNA Malat1 mRNA Levels in 3 Different Murine SkeletalMuscles

Wild type female CD-1 mice (approximately 6-8 weeks old) were dosed onceby a single IV bolus injection in the tail vein at 5 mL/kg body weight,of either Malat1 or Scramble AOCs, in which the siRNA was conjugated tomurine anti-Transferrin Receptor (mTfR1) antibody, at doses 0.3, 1, 3, 6mg/kg body weight (siRNA amount). Muscle tissue samples were collected 2weeks post treatment in tubes containing ceramic beads, flash frozen inliquid nitrogen, and then homogenized in 1 mL cold Trizol using aFastPrep-24 (MP Biomedicals). Homogenate supernatants were used for RNAisolation using Direct-zol-96 RNA isolation kit (Zymo) according to themanufacturer's instructions. 100-500 ng of purified RNA was converted tocDNA using High-Capacity cDNA Reverse Transcription Kit (AppliedBiosystems) using SimpliAmp Thermal Cycler (Applied Biosystems). cDNAwas analyzed by qPCR using TaqMan Fast Universal Master Mix II (ThermoFisher) and TaqMan probes (Thermo Fisher) in duplicates, usingQuantStudio 6 or 7 Flex Real-Time PCR instruments (Applied Biosystems).Data were analyzed by QuantStudio-Real-Time PCR Software v1.3 (AppliedBiosystems). The target gene expression was normalized to a referencegene Ppib. The percentage of target mRNA expression in treatment sampleswas determined relative to the control treatment using the 2^(−ΔΔCt)method. Data are represented as % of PBS control (mean±SEM; N=4 forsiMalat1 and siScramble AOCs, N=5 for PBS groups).

FIG. 19 shows Malat1 siRNA-AOC-mediated in vivo reduction of nuclearlocalized Inc-RNA Malat1 levels in skeletal muscles in mice. A singleadministration of up to 6 mg/kg of Malat1 siRNA-AOC (siRNA dose) in micereduced nuclear Malat1 expression up to 80% in skeletal muscle 2 weekspost-dose. The reduction of nuclear Malat1 mRNA expression levelsindicate the in vivo ability of AOCs to target nuclear RNAs fordegradation.

Example 13. Sustained AOC-Mediated In Vivo Reduction of SSB mRNA Levelsin Murine Skeletal Muscles with a Single Dose of 3 mg/kg siRNA AOCDuring an 8-Week Period

Wild type male C57BL/6 mice (approximately 12-16 weeks old) were dosedonce by a single IV bolus injection in the tail vein at 5 mL/kg bodyweight of either without vinylphosphonate (Non-VP) or withvinylphosphonate (vpUq) Ssb siRNA conjugated to murine anti-TfR1 (mTfR1)antibody at 3 mg/kg body weight (siRNA amount) dose. Gastrocnemiusmuscles were collected at the following time-points after dosing: day 1,7, 14, 28, 43 and 57. Muscles were placed in tubes containing ceramicbeads, flash frozen in liquid nitrogen, and then homogenized in 1 mLcold Trizol using a FastPrep-24 (MP Biomedicals). Homogenatesupernatants were used for RNA isolation using Direct-zol-96 RNAisolation kit (Zymo) according to the manufacturer's instructions.100-5(0) ng of purified RNA was converted to cDNA using High-CapacitycDNA Reverse Transcription Kit (Applied Biosystems) using SimpliAmpThermal Cycler (Applied Biosystems). cDNA was analyzed by qPCR usingTaqMan Fast Universal Master Mix 11 (Thermo Fisher) and TaqMan probes(Thermo Fisher) in duplicates, using QuantStudio 6 or 7 Flex Real-TimePCR instruments (Applied Biosystems). Data were analyzed by QuantStudio™Real-Time PCR Software v 1.3 (Applied Biosystems). Ssb gene expressionwas normalized to the reference gene Ppib. The percentage of target mRNAexpression in treatment samples was determined relative to the controltreatment (PBS) using the 2^(−ΔΔCt) method. Data are represented as % ofPBS control (mean f SEM; N=4 for siSsb-AOCs. N=3-5 for PBS groups).

FIG. 20 shows SSB siRNA-AOC-mediated in vivo reduction of SSB mRNAlevels in murine skeletal muscles with a single dose of 3 mg/kg siRNAduring an 8-week period. The sustained AOC-mediated in vivo reduction ofSSB mRNA expression levels was achieved in a mouse skeletal muscle aftera 3 mg/kg (siRNA dose) single administration of both withoutvinylphosphonate (non-VP) and with vinylphosphonate (vpUq) SSBsiRNA-AOCs.

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the disclosure. It should beunderstood that various alternatives to the embodiments of thedisclosure described herein may be employed in practicing thedisclosure. It is intended that the following claims define the scope ofthe disclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

What is claimed is:
 1. A method of delivering an oligonucleotide to asubject, wherein the subject has facioscapulohumeral muscular dystrophy(FSHD), the method comprising intravenously administering to the subjecta conjugate that comprises an anti-transferrin receptor antibodycovalently linked to a 5′ end or a 3′ end of an oligonucleotide, whereinthe oligonucleotide comprises one or more modifications and a strandthat comprises a region of complementarity of at least 15 nucleotides inlength to the nucleotide sequence of DUX4 mRNA; wherein theoligonucleotide is in the range of 15-35 nucleotides in length, whereinthe one or more modifications comprise a 2′-modified nucleoside selectedfrom the group consisting of: a 2′-O-methyl nucleoside, a 2′-fluoronucleoside, a 2′-O-methoxyethyl nucleoside, and 2′4′-bridgednucleosides, and combinations thereof, and/or comprise a modifiedbackbone selected from a backbone comprising one or morephosphorothioate linkages and a phosphorodiamidate morpholino backbone;and wherein the oligonucleotide brings about degradation of DUX4 mRNAand one or more DUX4 biomarker RNA in the muscle cell and wherein theone or more DUX4 biomarker RNA is selected from MBD3L2, TRIM43, PRAMEF1,ZSCAN4, KHDC1L, and LEUTX.
 2. The method of claim 1, wherein the regionof complementarity is at least 20 nucleotides in length.
 3. The methodof claim 1, wherein the oligonucleotide is covalently linked to a lysinein the anti-transferrin receptor antibody via a cleavable linker.
 4. Themethod of claim 3, wherein the cleavable linker comprises avaline-citrulline sequence.
 5. The method of claim 4, wherein thecleavable linker further comprises one or more poly ethylene glycolunits.
 6. The method of claim 1, wherein the anti-transferrin receptorantibody is in the form of a ScFv, Fab fragment, Fab′ fragment, F(ab′)2fragment, or Fv fragment.
 7. The method of claim 1, wherein theanti-transferrin receptor antibody is in the form of a Fab fragment. 8.The method of claim 1, wherein the subject has one or more deletions ofD4Z4 repeats in chromosome
 4. 9. The method of claim 8, wherein thesubject has 10 or fewer D4Z4 repeats.
 10. The method of claim 1, whereinthe muscle cell is a skeletal muscle cell, a cardiac muscle cell, or asmooth muscle cell.
 11. The method of claim 1, wherein the subject ishuman.
 12. A method of delivering an oligonucleotide to a subject,wherein the subject has a muscular dystrophy associated with mutation ofa repeat region, the method comprising intravenously administering tothe subject a conjugate that comprises an anti-transferrin receptorantibody covalently linked to a 5′ end or a 3′ end of anoligonucleotide, wherein the oligonucleotide comprises one or moremodifications and a strand that comprises a region of complementarity ofat least 15 nucleotides in length to an RNA encoded by a gene associatedwith the muscular dystrophy; wherein the mutation of the repeat regionresults in aberrant gene expression, wherein the oligonucleotide is inthe range of 15-35 nucleotides in length; wherein the one or moremodifications comprise a 2′-modified nucleoside selected from the groupconsisting of: a 2′-O-methyl nucleoside, a 2′-fluoro nucleoside, a2′-O-methoxyethyl nucleoside, and 2′,4′-bridged nucleosides, and/orcomprise a modified backbone selected from a backbone comprising one ormore phosphorothioate linkages and a phosphorodiamidate morpholinobackbone; and wherein the oligonucleotide brings about degradation ofthe RNA encoded by the gene associated with muscular dystrophy in themuscle cell,
 13. The method of claim 12, wherein the anti-transferrinreceptor antibody comprises a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2,and a LCDR3 of an antibody listed in Table 1 and Table
 2. 14. The methodof claim 12, wherein the muscular dystrophy is facioscapulohumeralmuscular dystrophy.
 15. The method of claim 12, wherein the musculardystrophy is myotonic dystrophy.